INTRODUCTION:
Ultrasonication induces particle agitation in a liquid through cavitation, forming microbubbles that oscillate with fluctuating pressure. These bubbles, undergoing rectified diffusion, reach resonance size during compression. Upon reaching desired content, they violently collapse within one acoustic cycle, generating high temperature, microjets, shock waves, microconvection, and microturbulence1,2. High-intensity ultrasonication disperses liquids and fragments materials effectively, while low-intensity ultrasound produces steady cavitation, enhancing convective transport for potential interaction with liquid and bacterial surfaces3.
Planktonic bacteria exhibit greater sensitivity to antimicrobial agents compared to their biofilm counterparts, attributed to metabolic and gene expression changes during sessile growth4,5. Biofilm shields bacteria from antimicrobials by adhering to drugs and impeding antibiotic transport6. "Persister" and "metabolically inactive" cells within biofilms resist conventional antibiotics, contributing to treatment inefficacy7. Planktonic bacteria, propelled by radiation pressure toward oscillating bubbles, experience rotational motion with elevated shearing rates8. This induces micro-streaming effects, loosening cell bunches, enhancing nutrient/waste diffusion, and improving productivity9. In the case of Escherichia coli (E. coli), a prominent pathogen, rising antimicrobial resistance poses a significant global health threat, prompting its designation as a priority pathogen by the World Health Organization10.
Norfloxacin, a fluoroquinolone derivative with potent bactericidal properties, has a chemical structure of 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid9,11. It binds near tyrosine-122 in the GyrA subunits of DNA gyrase, forming a drug-enzyme-DNA complex that inhibits DNA processes like supercoiling and relaxation12. DNA gyrase comprises two prokaryotic subunits, GyrA and GyrB, each with distinct functions encoded by gyrA and gyrB genes13. Fluoroquinolones specifically target the GyrA subunit, influencing cellular processes without affecting gyrase B or DNA directly. Factors affecting drug penetration into the cell are crucial for efficacy14. Various studies on the impact of biofilms on drug transport have demonstrated that they can occasionally impede but rarely entirely block drug transport7. Based on these findings, this current study focused on the following aspects: (1) the effect of low-intensity ultrasound on growth; (2) the synergistic effect of ultrasound and norfloxacin across Escherichia coli biofilms; (3) affinity prediction between the norfloxacin with pharmacological target (DNA gyrase subunit A) using computational methodology.
MATERIALS AND METHODS:
1. In vitro study:
Cultures of planktonic E. coli (MTCC-1427) were cultivated in tryptic soy medium. The minimum inhibitory concentration (MIC) of norfloxacin was assessed across a range of concentrations (0.5-100 μg/ml) using 96-well cell culture plates. The results were analyzed using a plate reader (Biorad iMark 14876; Japan). Subsequently, for additional experiments, norfloxacin concentration discs within the aforementioned range were prepared by loading 50μl of each desired solution onto blank discs made of Whatman's filter paper15.
1.2 Biofilm thickness estimation:
Biofilms were generated from cultured bacteria utilizing a semi-quantitative assay16. Borosilicate glass tubes, each weighing and measuring equally, were employed for uniform biofilm formation, with ultrasonic (US) transmission through the tube bottoms. For the biofilm formation process, an overnight culture of E. coli strain was cultivated in TSB medium and standardized according to the 0.5 MacFarland Standard. The inoculation into tubes containing 3ml of growth media was followed by a13-hour incubation at 37°C. A norfloxacin concentration of 50µg/ml, approximately five times higher than the lowest bactericidal dose for E. coli planktonic cells (10µg/ml), was employed. The acoustic intensity for the experiment was set at 22 and 33kHz (pulse on-off mode for 20sec:10sec), maintaining a constant temperature of 25°C (VCX500; Sonic and Material.Inc; USA). Biofilms in the test tubes underwent four distinct treatments: (i) Control group (not treated), (ii) Norfloxacin treatment, (iii) US exposure (15:30:45:60 min) only, and (iv) Norfloxacin combined with US exposure (15:30:45:60min). This experimental setup was replicated thrice, and all tubes were further incubated at 37°C for 6hours before subsequent analysis.
1.3 Viable count:
The viable bacteria in the biofilm and supernatant were counted in CFU. the biofilms were carefully scraped from the tube bottom and digested with 0.5% trypsin before being extensively vortexed in TSB. Serial dilution of the samples to 100-2 in PBS before plating on TSB agar and cultivated for 24hr at 37°C. The amount of CFU was calculated and represented concerning the test tube (CFU/cm2) (equation. 1) for bacteria that generate biofilms and the volume of TSB for planktonic cells (CFU/ml) (equation.2). This experiment was replicated thrice.
CFU/ cm2, the number of colonies formed per cm2 of surface,
so first working out the surface area of the test tube a
Radius is 1/2 of the diameter, so 
*t2 (t= radius of the test tube) which
comes X cm2
Tm2 (T= Test tube diameter) = Tcm2
Tcm2 * X cm2 = Y (Room surface)
N (no. of colonies) * Y = Z (colonies in t m2)
Z/ t m2 = CFU/m2 or CFU/cm2. Equation.1
Log (CFU/ml) = log10 CFU/ [(dilution factor * aliquot)] Equation.2
1.4 Changes in biofilm morphology:
E. coli strains were subjected to 200-2 dilution in TSB medium and subsequently incubated at 37°C for 13 hours to facilitate the formation of biofilms. Consistent with previously established protocols, test tubes containing the treated biofilms underwent a triple wash with 200µl of PBS. Subsequently, the biofilms were subjected to staining using 2% crystal violet for 5 minutes, followed by rinsing with double-distilled water and air-drying. Visualization and documentation of the biofilms were conducted utilizing a microscope (ULH100HG; Olympus, Japan), and images were captured using a Micropublisher 5.0 RTV. Each experimental condition was replicated in triplicate to ensure robustness and reliability of the results.
1.5 Diffusion experiments:
The experimental design employed in this study was modeled after the methodology outlined by Anderl17. Membrane filters (GSWPO4700; MF-Millipore) with a diameter of 25mm were positioned on nutrient agar plates, and a 10μl inoculation culture was introduced at the center. Subsequently, the filters were incubated at 37°C for durations of 13 and 24hours. Following incubation, the filters, now hosting biofilms, were transferred to other nutrient agar plates containing 100 µg/ml of norfloxacin. A blank concentration disc, positioned atop the biofilm on the upper filter, was covered by a 13-mm membrane filter and moistened with 40µl of TSB. The plates underwent exposure to 33 kHz ultrasound for durations of 45 and 60 minutes, as well as a sham ultrasound exposure. A fixture, connected to an ultrasonic bath, ensured the undisturbed posture of the plates throughout the experiment, allowing the norfloxacin plates to float on the water's surface. After the designated exposure times, concentration discs were extracted from the upper filter, placed on sterile polystyrene Petri plates, and cooled. Any discernible changes to the biofilm resulting from ultrasound exposure were examined. The exposed discs were subsequently compared to standard concentration discs of norfloxacin for further analysis.
2. In Silico Analysis:
Receptor proteins, topoisomerase subunit A proteins 1ZVU (79.77 kDa) and 1KZN (23.29 kDa) from E. coli, were extracted from the PDB database. Prankweb facilitated visualization of the binding sites for these proteins and their ligands. Autodoc Vina 1.1.2 was applied to eliminate residues, enhancing structural optimization18. The 3D structure of norfloxacin was obtained from PubChem (ID 4539), and both receptor and ligand were converted into pdbqt files. Pymol 2.5 was employed to introduce polar hydrogen atoms and Kollman charges, with subsequent removal of water molecules for refined analysis19,20. Molecular docking between E. coli GyrA and norfloxacin revealed binding affinities, using PyRx's Autodock Vina. The docked position with the lowest binding energy was selected. and ChimaeraX and Discovery Studio for visualizing the interaction between the active ingredient and the target protein21,22.
RESULT:
Biofilm antimicrobial susceptibility test:
For a 13 h planktonic E.coli, MIC was 10μg/ml. On developing in a biofilm mode, a 13 h bacterial biofilm showed a significant resistance to norfloxacin at a similar concentration to the planktonic counterpart (figure 1A).
Figure 1: (A) MIC determination of E.coli. (B) Viability of E.coli in Biofilm.
Recovery of viable E. coli from the biofilm and planktonic growth to evaluate the antibacterial efficacy of norfloxacin coupled with US. In the norfloxacin-treated group, the feasible bacteria CFU/cm2 recovered from biofilm (8.81E+03) was slightly lower than of control group (1.05E+04) but a much lower number is achieved on comparing with US + norfloxacin exposure (15, 30, 45 and 60min) for 22 kHz (3.60E+03, 3.20E+03, 1.87E+03, 1.07E+03) and 33 kHz (33.47E+03, 2.67+03, 1.47E+03, 8.14+03). Viable CFU/cm2 from the biofilms expressed in the log10 numbers for the 15 and 30 min US-only-treated group were found to be 1.39E+04, 1.21E+04 (22 kHz), and 1.60E+04, 1.69E+04 (33 kHz) respectively, which differ significantly from all control groups. There was a significant increase in the numbers of viable CFU/cm2 in the 15 and 30 min only US group, a declining number is observed in the later group 45 and 60 min, and a drastic decline is found along with the synergistic (norfloxacin + US) group (figure 1B). The amounts of CFU/ml of viable cells in the biofilm findings were comparable to those in planktonic bacteria. A microscopic inspection was done to validate the effectiveness of norfloxacin + US against bacteria in biofilms (figure 2A). The US group was found to have small micropores, in contrast to the control group, norfloxacin-treated group, and group that got 15 minutes of US therapy, all of which had packed and thick biofilm. The biofilm treated with US for more than 30 minutes, with or without antibiotics, had many micropores. A synergistic effect of ultrasound and antibiotics results in a completely distorted biofilm. Larger micropores were found exhibiting in these (figure 2B).
Biofilm growth was found to consistently decline when grown in average incubation, but an elevated increase was observed when exposed to ultrasound for 15 and 30 min. Beyond 30 min of US exposure, the growth was observed comparatively less as of ordinary incubated without. The experiment was justified by staining biofilm with crystal violet and taking preliminarily observation with the naked eye. E. coli biofilm thickness growing after synergistic effect (ultrasound+ norfloxacin) had an average concentration of 3.07E+02 CFU/mL for 22 kHz and 2.73E+02 CFU/mL for 33 kHz. The average concentration without ultrasound was 8.85E+02 CFU/mL. These are significant distinctions.
Figure.2 (A) Thick Biofilm formation: a (sham); b (Norfloxacin); c and d = (ultrasound for15 and 30 min).; Moderate Biofilm formation: e (ultrasound for 45 and 60 min); Weak Biofilm formation : f and g (ultrasound synergistic with Norfloxacin); (B) a (Sham); b (Norfloxacin); c (Ultrasound 15 and 30 min); d (Ultrasound 45 and 60 min); e (Ultrasound + Norfloxacin).
Diffusion experiment:
Overall, the findings demonstrated that the age of the biofilm considerably reduced the amount of antibiotic transported into the disc (figure 3). Most notably, unlike the non-insonicated discs, the discs above ultrasonicated biofilms contained more norfloxacin. There is a correlation between intensity and transport. When the biofilms were subjected to ultrasound for 45–60 minutes, they held considerably more norfloxacin than when utilizing non-insonated biofilms. When compared to exposure to 24hr biofilm, 33 kHz caused the transfer of more antibiotics for E. coli 13hr biofilm. A significant difference is noted that even at 45 min of sonication, the disc could not absorb more antibiotics from the 24hour biofilm. No sham treatment on 13 and 24hour biofilm transport of antibiotics showed similar results. Disk could not absorb antibiotics from biofilm at the speculative time; a higher contact time is needed for the absorbance in 13hr biofilm, but no effect was found in 24hr biofilm. Thus, it is concluded that insonation intensity enhances the transport of materials across the membrane. There appears to be a correlation between ultrasonic power and transbiofilm transfer volume. These transport methods are based on oscillatory accelerated diffusion, microconvection, and cavitation events.
Figure.3 Plate 1: Disc exposed to 24 hr Biofillm; Plate 2: Disc exposed to 13 hr Biofillm; Plate 3: Disc exposed to Biofillm without sham.
1 (Direct antibiotic Plate Exposed); 2 (Norfloxacin 10µg); 3 (60 min sonication exposed); 4 (45 min sonication exposed); 5 (24 hr biofilm); 6 (13 hr biofilm exposure for 240 min); 7 (13 hr biofilm exposure for 360 min); 8 (24 hr biofilm exposure for 360 min).
Docking:
Utilizing the outcomes of molecular docking, the scoring function was employed to compute the enthalpy's impact on the free binding energy value (Affinity ΔG) corresponding to the optimal conformational placements. The evaluation yielded free binding energy and binding constants (ΔGDoc kcal/mol and Ki (uM micromolar)) for specific ligand conformations, thereby aiding in assessing the stability of the formed complexes between the ligands and their respective targets. For each complex, nine conformations were found with different binding affinity. The lower the value of binding affinity, the better the docking. The docking analysis unveiled a heightened binding affinity between the molecule and its receptor. This interaction displayed spontaneity, enabling the drug to bind to the protein with a favorable orientation at a low concentration. This observation underscores the drug's potent efficacy in inhibiting bacterial gyrase activity. Notably, for the proteins 1zkn and 1zuv, the interaction residues exhibited values of -7.097 and -7.493 kcal/mol, respectively (figure 4A). It's important to note that the residues engaged in interactions with the optimal ligand conformation exhibit significant variability across different species. 1zkn and norfloxacin share interaction in residue ILE78, ASP73, THR165, ALA47, VAL71, VAL167, VAL130 through the hydrogen bond. 1zuv and norfloxacin share interaction in residue LEU257, HIS259, VAL261, GLN280, PRO294, THR295, and LEU68 through the hydrogen bond (figure 4B).
Figure. 4 (A). Image obtained using pymol showing the docking result of target with norfloxacin (a) 1zkn ; (b) 1zuv. (B). Interaction of the molecule with the residue of 1zuv and norfloxacin obtained after docking through autodockvina. (a) 1ZKN, (b) 1ZUV.
DISCUSSION:
At lower intensity and frequency ultrasonication, biofilms form more robustly, contradicting the misconception that ultrasound hinders their development. Bacteria still adhere to surfaces under ultrasound exposure. The observed effect arises from the intensity and duration of ultrasound, with studies using more potent ultrasound than that employed in the current study (22 and 33 kHz). Ultrasonic promotion of biofilm development may be linked to accelerated mass transfer, as demonstrated in various investigations hypothesizing23. Exopolymer mass transfer barriers in biofilms hinder bacterial growth. Ultrasound was expected to accelerate the movement of tiny molecules inside biofilms using mathematical models24,25. Various Investigations show that ultrasound at 100 W/cm2 at 40 kHz or 2 W/cm2 at 70 kHz dramatically improved mass transfer across biofilms26. The current experiments aimed to validate the theory that ultrasound, synergized with antibiotics, reduces bacterial biofilm load by enhancing antibiotic transport27. Mature biofilms, resilient to high drug concentrations, pose a challenge for conventional (MIC). Tests explored norfloxacin transport with and without ultrasound, hypothesizing sonoporation's role in amplifying antibiotic impact28. Even after a decade of research, a thorough understanding of sonoporation and its complete spectrum of bioeffect mechanisms remains largely elusive. Ultrasound-assisted drug transmission extends beyond bacterial membranes to the biofilm's extracellular matrix, influenced by glycopeptides and exopolysaccharides contributing to the physical hindrance of norfloxacin29. Results demonstrate ultrasound-mediated disruption of biofilm thickness and density. Longer exposure and low intensity are crucial, with 33 kHz outperforming 22 kHz. Extended synergism periods further s diminish biofilms, highlighting the method's potential efficacy. To our knowledge, This study is the first to explore how low-frequency ultrasound affects norfloxacin's antibacterial efficacy on in vitro biofilm cells. Combining antibiotics with ultrasound enhances antibacterial action against biofilms. Norfloxacin and ultrasound-treated biofilms exhibit the highest bacteria eradication, significantly reducing density with larger micropores. Ultrasound's impact involves increased cell permeability30,31, accelerated antibiotic transport, elevated temperature, intracellular production32, and reactive oxygen species permeabilizing cell membranes33. Larger micropores enhance antibiotic delivery, potentially aided by microbubbles acting as cavitation nuclei34,35. A multitude of research endeavors have proposed that ultrasound (US) could augment the mobility of genes or drugs within eukaryotic cells36. The phenomenon of reparable sonoporation discerned through scanning electron microscopy, has definitively elucidated this process37. While ultrasound's impact on eukaryotic cells is well-documented, further investigation is needed for bacterial walls due to size and structural differences.
Qian etal.'s research suggested that low-intensity ultrasound (10mW/cm2, 2 h) may enhance drug action against biofilms without disturbing or transferring bacteria38. However, our study revealed micropores in biofilms treated with ultrasound (US) and norfloxacin, indicating potential disturbance. The longer, higher-intensity US exposure might explain these results, aligning with findings from 0.07 MHz US after 45 minutes of exposure30. US-mediated antibiotic effectiveness on bacterial biofilms is influenced by acoustic strength39, duty cycle, frequency, and duration. Higher energy and duration can aid antibiotic transmission7. Biofilm age and bacterial type also affect antibiotic action28. Carmen 40reported that a 24-hour coupling of gentamicin with ultrasonic irradiation reduces E. coli biofilm persistence in vivo, indicating that such treatment decreased viable for 48hours to nearly zero. Although Pseudomonas aeruginosa biofilms were treated with US for 24 and 48hr, they had no appreciable reduction. The scientists hypothesized that the difference in responses between these two creatures was caused by the extended period, which increased permeability and decreased the stability of the outer membrane. Additionally, biofilm age significantly hinders antibiotic delivery31,41, emphasizing the importance of timely intervention for optimal outcomes. Generally, any biofilm inhibits significant antibiotic delivery. Notably, ultrasonicated biofilms exhibited higher norfloxacin content than non-insonated ones, indicating improved drug transfer despite biofilm age-related challenges. Norfloxacin penetration into the biofilm increased with prolonged exposure, allowing accumulation within the disc. Distinctions in physical, chemical, and physiological attributes were observed between biofilms developed over 13 and 24 hours, impacting antibiotic delivery. Regarding biological variations, Sauer et al. showed that genetic expression changes across several phases of biofilm development42. DNA gyrase, a bacterial Gyr component, makes it a potential drug target43. Norfloxacin, a fluoroquinolone, inhibits DNA gyrase subunit A, crucial for DNA replication. The fluoroquinolones primarily target gram-negative gyrase (topoisomerase II) and gram-positive topoisomerase-IV. However, the specific mechanism through which these result in the death of bacterial cells is yet unknown. The "two-gate model" has received substantial support from structural and biochemical data 44.
As biofilms grow, genetic expression changes affect antibiotic delivery. Various factors restrict drug dispersion in biofilms, including solid percentage, enzymatic breakdown, irreversible and reversible binding as demonstrated by Stewart et al.16. The fact that the age of the biofilm has a considerable impact on norfloxacin transport through the E. coli biofilm implies that one or more of Stewart's "retarding" characteristics become more pronounced as the biofilm grows. Antimicrobial resistance in older biofilms41 may involve binding, inactivation, or enzymatic destruction. In the absence of ultrasound, norfloxacin required 360 minutes for biofilm species effects in E. coli, whereas ultrasound-mediated penetration in a 24-hour-old biofilm took 240 minutes, without achieving antibiotic penetration even after 360 minutes of average incubation. Anderl et al.'s study on a wild-type and a β-lactamase-deficient mutant K. pneumoniae17 showed ampicillin failing to permeate wild-type biofilms within 240 minutes, while ciprofloxacin penetrated mutant biofilms in 10 minutes. Enzymatic breakdown hindered ampicillin penetration. Walters etal.45 reported tobramycin penetrating a P. aeruginosa colony biofilm after approximately 13 hours, exceeding our 45 minute examination. Antibiotic intrinsic chemistry crucially influences transport through biofilms.
CONCLUSION:
Exploring low-frequency ultrasound's potential for synergistic bactericidal effects is in early stages, demanding comprehensive investigation into crucial variables like bacterial composition and biofilm maturity. Calibration of ultrasound parameters is essential for engineering universally effective bactericidal impacts. Transitioning from lab to in vivo scenarios is imperative for understanding immune interactions. Animal studies are pivotal for evaluating ultrasound-microbubble-antibiotic interplay within in vivo biofilms, advancing therapeutic potential. Distinguishing human and bacterial cells at a microstructural level adds complexity. Scrutinizing differential responses to ultrasonic sonoporation is vital. The convergence of ultrasound and antibiotics offers a potent strategy against biofilms, promising revolutionary infection management and non-invasive, targeted treatments in patient care. In conclusion, delving into low-frequency ultrasound research holds transformative potential, driven by thorough investigation, parameter optimization, and cellular response delineation for innovative biofilm treatment and heightened antibiotic efficacy.
CONFLICTS OF INTEREST:
The authors have no relevant financial or non-financial interests to disclose.
COMPLIANCE WITH ETHICAL STANDARDS:
Human and animal rights: This article does not contain any studies with human or animal subjects.
AUTHORS’ CONTRIBUTIONS:
Sameer Ranjan Sahoo conducted the overall research and wrote the first initial draft. Utkalika Mallick conducted the bioinformatics analysis. Arun Kumar Pradhan reviewed the final manuscript, edited the initial draught manuscript, and oversaw the project.
ACKNOWLEDGMENTS:
The authors wish to express sincere thanks to Prof. (Dr.) Manoj Ranjna Nayak; President, S’O’A (Deemed to be University) Bhubaneswar, for providing the necessary facilities to carry out the investigation.
ABBREVIATIONS:
MIC Minimum Inhibitory Concentration
MBC Minimum Bactericidal Concentration
TSB Tryptic soy broth
PBS Phosphate buffered saline
LA LuriaBertine agar
Sham Non-ultrasound
CFU Colony Forming Unit
ml millilitre
Cm Centimetre
US Ultrasound
OD Optical density
Hr Hour.
NA Nutrient Agar
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Received on 12.09.2023 Modified on 14.01.2024
Accepted on 26.04.2024 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(9):4381-4388.