Cytotoxicity and antibacterial BioEffects of CuO-Zn nanoparticles synthesized from Lactobacillus helveticus yogurt isolates targeting RsbA gene expression

Abstract

Proteus mirabilis is an opportunistic urinary tract pathogen distinguished by its swarming motility and dimorphic behavior. This study examined the antibacterial potential of native Bulgarian dairy Lactobacillus strains against clinically significant P. mirabilis isolates. By developing and characterizing copper oxide (CuONPs) and zinc oxide (ZnONPs) nanoparticles as antimicrobial agents targeting P. mirabilis, this work contributes to addressing the global challenge of antimicrobial resistance, aligning with the United Nations Sustainable Development Goals (UNSDGs) that aim to reduce infectious disease mortality through innovative therapeutic approaches. Two multidrug-resistant P. mirabilis isolates (M1 and M2) displaying swarming motility were selected for analysis. The antimicrobial activity of Lactobacillus helveticus, a heterofermentative lactic acid bacterium commonly found in dairy products, was evaluated. This strain produces helveticin, a bacteriocin-like inhibitory substance (BLIS) with notable bactericidal activity. CuONPs and ZnONPs were biosynthesized using L. helveticus isolated from yogurt, with strain identity confirmed via 16 S rRNA gene sequencing. Nanoparticles were subsequently extracted and partially purified through 70% ammonium sulfate precipitation. Characterization revealed surface plasmon resonance peaks at 250 nm (CuONPs) and 300 nm (ZnONPs). AFM analysis indicated particle sizes ranging from 15 to 26 nm, while TEM imaging showed predominantly spherical to irregular morphologies between 9 and 20 nm. Cytotoxicity evaluation using the MTT assay demonstrated dose-dependent reductions in the viability of WRL68 human hepatic cells, with up to 60% inhibition at higher concentrations. Notably, both nanoparticle types effectively inhibited P. mirabilis swarming motility, and qPCR analysis confirmed significant downregulation of the rsbA gene, a crucial regulator of this behavior. Overall, green-synthesized CuONPs and ZnONPs derived from L. helveticus displayed potent anti-swarming and antivirulence effects against P. mirabilis. Their ability to suppress virulence-associated gene expression underscores their promise as eco-friendly antimicrobial agents for managing opportunistic pathogens and mitigating multidrug resistance.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-49023-y.

Introduction

Lactic acid bacteria (LAB) have long been employed in the food industry as starter cultures owing to their ability to produce a wide range of bioactive compounds, such as amines, bacteriocins, and short-chain fatty acids¹. Strains capable of generating bacteriocin-like substances have been identified under conditions where the effects of hydrogen peroxide and organic acids were eliminated, confirming that these compounds primarily drive the inhibitory activity against target microorganisms². For instance, antioxidant peptides isolated from cheddar cheese were shown to increase in abundance with the addition of Lactobacillus helveticus, demonstrating the strain’s ability to enhance bioactive peptide release².

Bacteriocins—ribosomally synthesized antimicrobial peptides—exert inhibitory against both Gram-positive and Gram-negative bacteria³. L. helveticus is widely recognized as an important source of bioactive peptides in yogurt production, and bacteriocins derived from this species can help stabilize probiotic cultures in fermented dairy products⁴. Certain bacteriocins, including those effective against methicillin-resistant Staphylococcus aureus (MRSA), act by disrupting bacterial cell membranes to release ions such as K⁺ and inorganic phosphate or by inhibiting protein, DNA, and RNA synthesis in a bacteriostatic manner⁵. Moreover, the integration of probiotics and plant extracts into food products introduces physiologically active microorganisms and phytochemicals, enhancing nutritional quality and providing natural preservation⁶.

Proteus mirabilis, an opportunistic pathogen associated with urinary tract infections (UTIs) and catheter-associated UTIs (CAUTIs), exhibits pronounced swarming motility that promotes biofilm formation, encrustation, and catheter blockage. These factors contribute to recurrent infections, heightened antibiotic resistance, and severe clinical complications. Targeting such virulence mechanisms represents a promising strategy for infection control and public health improvement. However, both Gram-positive and Gram-negative bacteria may display resistance to certain bacteriocins. Currently, bacteriocins such as subtilin, nisin, and pyocin are employed in food and medical applications due to their broad-spectrum antimicrobial activity⁷.

P. mirabilis possesses multiple virulence factors that facilitate infection across diverse body sites, including hemolysins, proteases, urease, and biofilm-forming capabilities—all of which are secreted into the extracellular environment and contribute to pathogenicity⁸. Phenotypic and genetic analyses of these virulence determinants are critical for understanding infection dissemination and developing effective clinical interventions^9,10.

Green synthesis approaches—such as using plant extracts and environmentally friendly microbial systems—have been increasingly applied to produce metal nanoparticles like copper nanoparticles (CuNPs). These techniques improve the biocompatibility and functional properties of nanoparticles while minimizing ecological impact¹¹.

Previous research with silver nanoparticles has shown that they can suppress the expression of swarming-related genes such as rsbA and flhD, directly affecting bacterial motility and virulence¹². Building upon this concept, the present study aimed to evaluate the phenotypic and genotypic anti-swarming effects of eco-friendly, green-synthesized CuONPs and ZnONPs against P. mirabilis. Suppression of swarming motility was closely linked to downregulation of the rsbA gene, indicating a targeted antivirulence mechanism. We propose that combining these nanoparticles with antibiotics could potentiate bactericidal or bacteriostatic effects while reducing cytotoxicity in human cells.

A distinctive feature of this study is the use of Lactobacillus helveticus for nanoparticle biosynthesis. Unlike many other microbial or plant-based systems, L. helveticus is a GRAS (Generally Recognized As Safe) microorganism extensively used in dairy fermentation, ensuring biosafety and translational feasibility for biomedical and food applications. Its intrinsic production of helveticin and other bioactive metabolites enhances the reducing and stabilizing potential of the system, likely improving nanoparticle uniformity, stability, and bioactivity.

Materials and methods

Fermented yogurt samples were obtained from commercial markets in Baghdad, Iraq, and transported to the laboratory under chilled conditions (4 °C). Samples were processed within 3–5 h of collection. Lactic acid bacteria (LAB) were selectively isolated using de Man, Rogosa, and Sharpe (MRS) agar (Solarbio) through the serial dilution and spread plate technique, At conditions at 37 °C for 24 h¹³.

Screening for bacteriocin-like inhibitory substances (BLISs)

Lactic acid bacteria (LAB) strains were cultivated in MRS liquid medium under anaerobic conditions at 37 °C for 24 h. Following incubation, the cultures were centrifuged at 5000 × g for 8 min at 4 °C to remove cellular debris. The resulting supernatant was filtered through a 0.22 μm membrane to obtain bacteriocin-like inhibitory substances (BLIS).

The antibacterial activity of the BLIS was assessed against Proteus mirabilis using the well diffusion method. Briefly, 100 µL of an exponential-phase P. mirabilis suspension (10⁷ CFU/mL) was evenly spread onto Mueller–Hinton agar plates, and 200 µL of BLIS was introduced into wells (8.0 mm diameter) cut into the agar. The diameters of the inhibition zones surrounding the wells were measured to determine activity¹⁴.

The LAB strain that exhibited the strongest antibacterial effect against P. mirabilis was selected for subsequent analyses. This strain was re-cultured in MRS liquid medium for 24 h at 37 °C. Molecular identification was then performed by extracting genomic DNA using a DNA Purification Kit, following the manufacturer’s protocol¹⁵.

Ammonium sulfate precipitation and dialysis

Ammonium sulfate [(NH₄)₂SO₄] at saturation 60% was gradually added to the sample with continuous stirring on ice to facilitate protein precipitation. The mixture was then centrifuged at 6,000 rpm for 20 min at 4 °C, and the resulting pellet was resuspended in phosphate buffer. The suspension was subsequently dialyzed using a 3,500 Da molecular weight cut-off membrane. After dialysis, enzyme activity and protein concentration were assessed. Overnight at 4 °C, 60% (NH₄)₂SO₄ saturation was used to precipitate the bacteriocin-like inhibitory substance (BLIS) from Lactobacillus helveticus culture supernatant. The precipitate was resuspended in 0.05 M phosphate buffer (pH 7.0), dialyzed against the same buffer, and collected by centrifugation. With a two-fold increase in specific activity and a recovery yield of roughly 50–60% of total antibacterial activity, the process demonstrated partial purification of a proteinaceous molecule compatible with helveticin-like BLIS.

Estimation of protein concentration

Protein concentration was determined using the Bradford assay, according to the method described by Bradford (1976)¹⁶.

Isolates

Two Proteus mirabilis strains, designated M1 and M2, exhibiting multidrug resistance and characteristic swarming motility, were obtained from the Department of Biology, College of Science, University of Baghdad, Iraq. These strains were originally isolated from patients with urinary tract infections and individuals with indwelling urinary catheters. For laboratory cultivation, the isolates were grown on MacConkey and blood agar plates at 37 °C for 24 h. Species identification was further verified using the Vitek-2 automated system¹⁷.Swarming motility assay.

Swarming motility was evaluated following previously described protocols^18,19. Briefly, LB swarm agar plates were inoculated on opposite sides with 5 µL of overnight Proteus mirabilis cultures and incubated at 37 °C for 20 h.

For culture revival, the P. mirabilis isolates (M1 and M2) were initially streaked onto 5% sheep blood agar (SBA) plates and incubated at 37 °C for 24 h. A single well-isolated colony from each plate was then suspended in sterile saline to achieve a turbidity corresponding to 10⁸ CFU/mL (0.5 McFarland standard).

For the swarming assay, 100 µL of the standardized bacterial suspension was inoculated at the center of freshly prepared SBA plates. The plates were incubated at 37 °C for 24 h, after which swarming motility was visually assessed. The diameter of the swarming zone was measured in millimeters using a ruler, and motility was recorded for each isolate based on the extent of bacterial surface expansion across the agar¹⁹.

16S rRNA gene sequencing

Molecular identification of Lactobacillus helveticus

The identification of Lactobacillus helveticus was carried out using 16 S rRNA gene sequencing. Depending on growth conditions, single colonies of each isolate were subcultured and incubated for 24–48 h. Genomic DNA was extracted using the ABIOpure™ Total DNA Kit (USA), following the manufacturer’s protocol. The 16 S rRNA gene (~ 1,500 bp) was amplified using the following universal primers:

• Forward: 5′-AGAGTTTGATCCTGGCTCAG-3′.

• Reverse: 5′-TACGGTTACCTTGTTACGACTT-3′²⁰.

PCR amplification was performed under the following conditions: an initial denaturation at 95 °C for 2 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s; followed by a final extension at 72 °C for 1 min.

Amplified PCR products were submitted The resulting sequences were analyzed using the BLAST database to confirm the taxonomic identity of the isolates based on sequence similarity.

Synthesis of nanoparticles

The selected bacterial strain, Lactobacillus helveticus H1, which produced a bacteriocin-like inhibitory substance (BLIS), was employed for the biosynthesis of metal oxide nanoparticles.

For zinc oxide nanoparticle (ZnONP) synthesis, 20 µL of 1 mM zinc acetate [Zn(O₂CCH₃)₂] was added to 5 mL of BLIS and incubated at room temperature in the dark for 24 h²⁰.

For copper oxide nanoparticle (CuONP) synthesis, 1 mL of 5 mM copper(II) acetate was mixed with 5 mL of BLIS and incubated under identical conditions—at room temperature in the dark for 24 h²¹.

Analysis of synthesized nanoparticles

To confirm the formation of nanoparticles, copper acetate and zinc acetate solutions were added to the BLIS, while a control sample without metal ions (C) was maintained for comparison (Fig. 1). The reduction of metal ions and subsequent nanoparticle synthesis were monitored using a UV–visible spectrophotometer across a wavelength range of 300–700 nm²². The appearance of characteristic surface plasmon resonance (SPR) peaks served as evidence of successful nanoparticle formation.

Fig. 1.

Steps of biosynthesis CuONPs and ZnONPs.

Characterization of the nanoparticles

The synthesized nanoparticles (NPs) were characterized to determine their size, shape, and structural properties. UV–visible spectroscopy (UV/Vis-1800, Shimadzu, Kyoto, Japan) was used to confirm nanoparticle formation²³. Functional groups present on the nanoparticles were identified using Fourier transform infrared spectroscopy (FT-IR, Bruker, Esslingen, Germany).XRD anylisis.

Morphological features were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM, Hitachi S-3400 N, Japan), while atomic force microscopy (AFM) was employed to assess surface topography. For analysis of small sample amounts, either smear slide mounts or powdered sample slurries were prepared²⁴.

MIC determination

The minimum concentration of CuNPs and ZnONPs that inhibited bacterial growth was calculated using Eppendorf tubes and the broth microdilution method. After being prepared in MHB, the concentration of the bacterial inoculum was adjusted to 10 ⁸ CFU/µl/µl. Six tubes were used to create twofold serial dilutions of CuO NPS (16, 32, 64, 128, 256, and 512 µg/µl) using MHB. The tubes were then incubated at 37 °C for a whole day. The next step was to identify any visible bacterial growth in each tube, which allowed for the determination of sub-MICs²⁵. Gentamicin (10 µg/mL) was used as a positive control to benchmark the antibacterial activity of CuONPs and ZnONPs.

Application studies

Anti-swarming effect of the MICs of the nanoparticles

Swarming motility assays were conducted with slight modifications. Test isolates were first incubated in Brain Heart Infusion Broth (BHIB) for 24 h at 37 °C. Subsequently, 5 µl from each culture was collected and diluted with PBS to achieve a concentration of 1.5 × 10⁸ CFU/ml. One milliliter of this diluted suspension was then co-incubated with 1 ml of ½ MIC concentrations of CuNPs or ZnONPs for 24 h at 37 °C. Following incubation, 10 µl of each mixture was inoculated at the center of pre-prepared 5% human blood agar (HBA) plates. Results were recorded, analyzed, and compared with those of the untreated (negative control) group to assess the impact of CuNPs and ZnONPs on bacterial motility²⁶.

MTT assay for cytotoxicity evaluation

The cytotoxicity of CuNPs and ZnONPs was evaluated using the in vitro 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 7,000 cells were seeded into each well of a 96-well plate and incubated overnight to allow for proper cell adherence. The WRL68 and normal A375 cell lines were then exposed to increasing concentrations of CuNPs and ZnONPs (0.5, 1.0, 1.5, and 2.0 µg/ml), with each treatment performed in triplicate. After 24 h of incubation, the culture medium was removed, and 20 µl of MTT solution (5 mg/ml; Shanghai Macklin Biochemical Co., Ltd.) was added to each well. The plates were incubated for an additional 3 h at 37 °C in the dark to allow for the formation of formazan crystals. Subsequently, 50 µl of dimethyl sulfoxide (DMSO; Bio Basic Inc.) The absorbance was measured at 490 nm²⁷.

Cell viability (%) was calculated based on absorbance values using the following formula:

Viability (%)=Atest−AblankAcontrol−Ablank×100\text{Viability (\%)} = \frac{A_{\text{test}} - A_{\text{blank}}}{A_{\text{control}} - A_{\text{blank}}} \times 100Viability (%)=Acontrol​−Ablank​Atest​−Ablank​​×100 (2).

Gene expression analysis of RsbA

To determine the effect of ZnONPs and CuONPs on gene expression, each bacterial isolate was suspended in Mueller–Hinton Broth and treated with sub-MIC concentrations of the nanoparticles on separate plates. The cultures were incubated overnight at 37 °C. Following incubation, the expression of the RsbA gene was analyzed to assess the regulatory impact of nanoparticle exposure. The rpoA gene served as a housekeeping gene and was used to normalize the ΔCt values of the target gene for accurate quantification.

RNA extraction

Total RNA was extracted from samples before and after treatment with the three nanoparticles using TRIzol™ reagent, following the manufacturer’s protocol. This ensured efficient lysis and purification of high-quality RNA suitable for downstream gene expression analysis.

RNA quantification

The concentration and purity of the extracted RNA or synthesized cDNA were assessed using a Quantus Fluorometer. One microliter of RNA or cDNA was mixed with 199 µl of diluted QuantiFluor Dye, followed by incubation for 5 min at room temperature in the dark. The fluorescence intensity was then measured to determine RNA concentration and assess sample quality for subsequent molecular analyses.

Gene expression analysis

Relative quantification was used to evaluate gene expression levels. Fold change variations and cycle threshold (ΔCt) values between the control (calibrator) and treated groups were calculated using the comparative Ct (2^−ΔΔCt) method, as described by Livak and Schmittgen (2001)²⁸. Gene expression levels in P. mirabilis were normalized against the housekeeping gene rpoA, ensuring accurate and reliable quantification of target gene (RsbA) expression.

Relative quantification

1

2

3

4

5

A melting curve was generated by gradually increasing the temperature from 72 °C to 95 °C at a rate of 0.3 °C/s to verify the specificity of amplification and the absence of primer-dimer formations.

Real-time PCR protocols

Real-time PCR amplification was carried out to confirm the presence and expression of the RsbA and rpoA genes in P. mirabilis. The process consisted of two main stages. In the first stage, complementary DNA (cDNA) was synthesized from total RNA using the ProtoScript cDNA Synthesis Kit, following the manufacturer’s guidelines. Gene-specific primers targeting RsbA and rpoA transcripts were used for reverse transcription. To prevent genomic DNA contamination, the RNA samples were treated with DNase I prior to cDNA synthesis, as recommended in the manufacturer’s protocol.

The primer sequences and amplification conditions used for each gene are listed in Table 1, and the corresponding real-time PCR reaction components and cycling parameters are summarized in Table 2.

Table 1.

Primers used in this study.

Primer Name		Sequence	Product size (bp)	Reference
rsbA	F	F 5- CTATACCTACCGCACCATGT-3	180	29
	R	5- GAAGTCCCATCCGTTGATAC-3
ropA	F	5-GCGTGTTATAGCCCAGTTGA-3	198	30
	R	5-AGGCTGACGAACATCACGTA-3

Table 2.

Synthesis of cDNA from RNA using primers for RsbA and rpo transcripts.

Component	Volume
Luna Universal Master Mix	10 ul
Forward primer	1 ul
Reverse primer	1 ul
Template DNA	5 ul
Nuclease-free Water	3 ul
Total volume	20 ul

The expression levels of the rsbA and rpoA genes were analyzed in the second stage of the experiment under the following thermal cycling conditions. The rpoA gene served as an internal reference for normalization. After centrifugation of the PCR tubes at 2000 × g for 1 min, the reaction mixture was briefly spun down before running the reactions. Real-time PCR software was used to acquire and analyze amplification data. Fold changes and cycle threshold (Ct) differences between the control (calibrator) and treated groups were calculated using the Livak (2^−ΔΔCt) method to determine relative gene expression levels.

Ethical statement

This research was approved by the Committee of Ethical Standards, College of Sciences, University of Baghdad. The study protocol, participant information, and consent form were reviewed and authorized under document number FM.SA/308, dated December 25, 2023.

Statistical analysis

All experiments were carried out in triplicate, and the results are expressed as mean ± standard deviation (SD). The effects of experimental treatments on biofilm formation and gene expression were evaluated using a Student’s t-test. Statistical analyses were performed using SPSS software version 26, and differences were considered statistically significant at P < 0.05.

Results

Serially diluted yogurt samples were plated on MRS agar and incubated for 48 h. Distinct bacterial colonies were then selected and recultured on fresh MRS agar plates for further analysis (Fig. 2).

Fig. 2.

(A) Growth of bacterial colonies cultured on MRS agar. (B) Microscopic examination.

Screening of isolated colonies

The antimicrobial activity of bacteriocin-like inhibitory substances (BLISs) produced by the isolated lactic acid bacteria (LAB) strains was assessed against selected bacterial pathogens. In particular, their inhibitory effects were evaluated against multidrug-resistant Proteus mirabilis strains M1 and M2, which had been previously confirmed using the Vitek-2 system (Fig. 3). The BLIS extracts exhibited pronounced antibacterial activity, producing inhibition zones of 20 mm and 21 mm, respectively. These results demonstrate the strong antagonistic potential of the LAB-derived BLISs against gram-negative P. mirabilis isolates.

Fig. 3.

Antibacterial activity of synthesized nanoparticles against Proteus mirabilis.

Determination of 16 S rRNA sequence identification (FLAB)

To genetically characterize the Lactobacillus helveticus isolate obtained from yogurt, the strain demonstrating the strongest inhibitory activity against multidrug-resistant Proteus mirabilis was selected for molecular identification. The 16 S rRNA gene was amplified using polymerase chain reaction (PCR), producing an amplicon of approximately 917 bp, as verified by comparison with a molecular DNA ladder (Figs. 4a and 5). This confirmed the successful amplification of the target gene for subsequent sequencing and phylogenetic.

Fig. 4.

Amplification results of the 16 S rRNA gene of Lactobacillus helveticus following separation on a 1.5% agarose gel and visualization with ethidium bromide staining. M: 1000 bp DNA ladder marker; Lane 5: PCR amplicon corresponding to the 917 bp.

Fig. 5.

NCBI nucleotide BLAST alignment results for the 16 S rRNA gene sequence of Lactobacillus helveticus. The query represents the amplified 16 S rRNA gene sequence from the isolated L. helveticus strain, while the subject corresponds to the NCBI reference strain of L. helveticus (Accession ID: NR_042439.1). The high sequence similarity confirms the molecular identification of the isolate as Lactobacillus helveticus.

Isolation and identification of P. mirabilis

Using the VITEK 2 automated system, which is recognized as a reliable method for bacterial identification and antimicrobial resistance profiling, two isolates were confirmed as multidrug-resistant (MDR) Proteus mirabilis (Fig. 6). The identification was further supported by characteristic colony morphology on MacConkey agar—showing pale, non-lactose fermenting colonies—and the distinctive swarming motility pattern observed on blood agar plates.

Fig. 6.

Vatic 2 system for P. mirabilis.

Swarming assay

The swarming motility of both Proteus mirabilis isolates was evaluated on blood agar plates following incubation at 37 °C for 24–48 h. The swarming zones were measured in millimeters, with diameters ranging from 15 mm for isolate M1 to 22 mm for isolate M2. P. mirabilis displayed the characteristic bull’s-eye swarming pattern, indicative of its strong motility and surface colonization ability. This behavior contributes to its capacity to migrate rapidly across solid surfaces, such as urinary catheters, thereby promoting infection dissemination within the urinary tract (Fig. 7).

Fig. 7.

Swarming motility of P. mirabilis (A) M1 (B) M2 isolates on blood agar at 37 °C for 48 h.

Biosynthesis nanoparticles

The potential of Lactobacillus helveticus BLIS for synthesizing metal nanoparticles was further investigated. Copper oxide nanoparticles (CuONPs) were identified based on a color change from yellow to blue and the formation of a light blue to white precipitate. For zinc oxide nanoparticles (ZnONPs), a precipitate formed after centrifugation, which was subsequently dried using a microwave to yield bright blue and white powders, representing the synthesized nanoparticles (Fig. 8).

Fig. 8.

Strictures of nanoparticles via L. helveticus (A) CuONP and (B) ZnONP powders.

Characterization of nanoparticles

UV–Vis spectral analysis

The results showed that the absorption peak at 250 nm was the maximum for the biosynthesized CuoNPs, while the peak at 300 nm was attributed to the ZnONPs, as shown in Fig. 9.

Fig. 9.

UV–Vis spectrophotometry spectrum of synthesized nanoparticles showing (A) CuONPs and (B) ZnONPs.

Atomic force microscopy (AFM) analysis

To determine and characterize the distribution of nanoparticles, AFM analysis was performed on both CuO and ZnO nanoparticles. As shown in Fig. 10, the grain sizes range between 13 and 26 nm. AFM was used to examine the surface morphology and roughness of the synthesized nanoparticles. The 3D topographic images display a relatively uniform particle distribution with distinct surface elevations. The measured surface height varied from 0 to 12 nm, confirming the nanoscale dimensions of the particles. Surface roughness analysis revealed a moderately rough texture, consistent with the agglomerated morphology observed in TEM and FESEM images. This nanoscale roughness may enhance surface reactivity and increase the number of active sites, offering potential benefits for biomedical applications such as drug delivery and antimicrobial activity.

Fig. 10.

3D AFM images of the (A) CuO NPs and (B) ZnOPs.

Transmission electron microscopy (TEM) analysis

The size and morphology of the nanoparticles were analyzed using TEM imaging. TEM observations confirmed the core structure of the synthesized nanoparticles. As presented in Fig. 11, the images display well-defined spherical particles with diameters ranging from 13 to 29 nm.

Fig. 11.

Transmission electron microscopy (TEM) their spherical morphology: (A) CuO NPs and (B) ZnO NPs.

Field emission scanning electron microscopy (FESEM) and XRD

SEM analysis was conducted to evaluate the morphology, size, and elemental as well as structural composition of the nanoparticle samples. As shown in Figure (12, 13) the ZnO NPs and CuO NPs exhibit predominantly spherical shapes at a magnification of 3000 kV. The SEM images revealed particle diameters ranging from 15 to 26 nm, confirming their essentially spherical morphology.

Fig. 12.

Scanning electron microscopy (SEM) images of the nanoparticles showing their spherical morphology: (A) ZnO NPs and (B) CuO NPs.

Fig. 13.

XRD : (A) CuO NPs and (B) ZnO NPs.

Minimum inhibitory concentration (MIC) of CuO NPs and ZnO NPs

The minimum inhibitory concentrations (MICs) of CuO and ZnO nanoparticles were evaluated against both P. mirabilis isolates (M1 and M2). Complete inhibition of bacterial growth was observed at 64 µg/mL for both CuO and ZnO nanoparticles. Sub-MIC concentrations were then employed to assess their impact on swarming motility and gene expression.

Anti-swarming activity of sub-MIC CuO and ZnO NPs

The two swarming P. mirabilis isolates were exposed to sub-MIC concentrations of the biosynthesized CuO and ZnO nanoparticles. Both nanoparticles markedly inhibited swarming activity after 24 and 48 h of incubation at 37 °C. A significant decrease in colony diameter was observed for both isolates. Notably, isolate M2 exhibited a swarming diameter of 22 mm on SBA plates before treatment, which was completely suppressed following exposure to either nanoparticle (Fig. 14).

Fig. 14.

Swarming diameter after treatment with (A) CuO NPs and (B) ZnO NPs for P. mirabilis M2 isolates.

Cytotoxic effect

The MTT assay was employed to evaluate the cytotoxic and antitumor activities of the biologically synthesized CuO and ZnO nanoparticles against the WRL68 (normal hepatic) and A375 (melanoma) cell lines. The results showed a clear dose-dependent decrease in cell viability, indicating that higher nanoparticle concentrations led to stronger cytotoxic effects—much like increasing the intensity of sunlight gradually wilting a delicate leaf.

For CuO NPs, the IC₅₀ value against WRL68 cells was approximately 7.367 µg/mL, with a maximum growth inhibition of 60%. Against A375 cells, the inhibition reached only 20%, which was insufficient to determine a reliable IC₅₀ (Fig. 15).

Fig. 15.

Cell survival curves of (A) CuONPs and (B) ZnONPs.

For ZnO NPs, the IC₅₀ values were approximately 152.4 µg/mL for A375 cells and 120.7 µg/mL for WRL68 cells. These findings highlight the dose-dependent cytotoxicity of both nanoparticles and their selective activity toward normal hepatic (WRL68) versus malignant melanoma (A375) cells—similar to how certain keys fit specific locks, these nanoparticles interact differently depending on the cell type they encounter (Fig. 15).

Fig. 16.

Expression of the rsbA gene in P. mirabilis is significantly downregulated following treatment with CuO-NPs. Different letters above the bars (a, b) denote a statistically significant difference (P < 0.001).

RsbA gene expression

P. mirabilis after treatment with metal oxide nanoparticles (P < 0.05). Relative to untreated controls (set at 1), CuO-NPs reduced rsbA expression to 0.52-fold, while ZnO-NPs caused a more substantial reduction to 0.32-fold. Statistical analysis confirmed that the suppression by ZnO-NPs was significantly greater than that by CuO-NPs (P < 0.05), demonstrating a differential impact of nanoparticle type on gene expression (Fig. 18).

Fig. 18.

Comparative effect of CuO-NPs and ZnO-NPs on rsbA gene expression in P. mirabilis. Both treatments caused a significant downregulation compared to the untreated control, with ZnO-NPs exhibiting a stronger suppressive effect than CuO-NPs. Bars labeled with different letters (a, b, c) are statistically distinct (P < 0.001; n = [number]).

Fig. 17.

Treatment with ZnO-NPs significantly reduced the expression of the rsbA gene in P. mirabilis. Bars labeled with different letters (a, b) indicate a statistically significant difference (P = 0.006; n = [number]).

Discussion

Bacteriocins are promising alternatives to conventional antibiotics in the face of persistent bacterial infections. Their potential extends beyond antibacterial activity to include antioxidant and anticancer effects. Our findings confirm previous reports on the strong antimicrobial activity of Lactic Acid Bacteria (LAB), primarily attributed to bacteriocin production³¹.

The efficacy of bacteriocins is influenced by environmental conditions. For instance, the antagonistic effect of Lactobacillus species is enhanced in acidic environments, with optimal activity around pH 6³². However, naturally occurring bacteriocins face significant limitations, including modest activity against pathogens, rapid degradation, low yield, and a pH-dependent efficacy that declines as pH rises^33,34.

Nanotechnology offers a powerful solution to these challenges. Metabolites from bacteria like Lactobacillus helveticus can themselves be used to synthesize stable nanoparticles, which act as excellent platforms for bacteriocin delivery³⁵. By encapsulating bacteriocins, nanoparticles can enhance their stability, bioavailability, and therapeutic potential, overcoming the barriers that have limited their clinical application³⁶.

The combination of bacteriocins like helveticin with nanometal oxides (e.g., CuO and ZnO NPs) presents a strategy for synergistic antibacterial activity. Bacteriocins weaken bacterial defenses by disrupting cell membranes and interfering with essential processes. Simultaneously, nanoparticles induce oxidative stress and structural damage. Together, bacteriocins may sensitize cells and enhance nanoparticle penetration, leading to a combined effect greater than the sum of their individual actions.

In this study, we synthesized copper and zinc nanoparticles using a bacteriocin-like inhibitory substance (BLIS) from Lactobacillus helveticus and compared the bioactivity of the pure bacteriocin to its nanoparticle formulations. The nanoparticles exert their effect by generating reactive oxygen species (ROS), which cause oxidative stress, compromise membrane integrity, and lead to cell death. Their high surface area and stability in biological fluids further enhance this efficacy^37,38. Thus, formulating bacteriocins into nanoparticles represents a promising approach to amplify their antimicrobial properties³⁹.

The characteristic “bull’s-eye” swarming of Proteus mirabilis, mediated by its peritrichous flagella, is a key virulence factor that promotes catheter and urinary tract colonization⁴⁰. To target this specific behavior, we formulated bacteriocin-incorporated CuO and ZnO nanoparticles. These formulations demonstrated superior efficacy compared to the bacteriocin (BLIS) alone, significantly inhibiting swarming motility and indicating a targeted disruption of this critical virulence mechanism⁴¹.

This approach is particularly relevant for clinical isolates of P. mirabilis, where virulence genes are strongly correlated with swarming ability and pathogenicity⁴². The isolates used in this study, selected from hospital-acquired urinary tract infections based on unique susceptibility profiles, underscore the connection between virulence factors, bacterial dissemination, and antibiotic resistance.

Beyond their established antibacterial role, bacteriocins and nanoparticles show significant promise in anticancer therapy. As conventional treatments like chemotherapy and radiation are often plagued by severe side effects, there is a growing impetus to explore alternative natural agents, including bacteriocins and biosynthesized nanoparticles⁴³. Modern drug discovery tools, such as molecular docking, provide a cost-effective framework to validate the therapeutic potential of these compounds by predicting their interactions with specific cellular targets⁴⁴.

In summary, this study underscores the dual functionality of bacteriocin-based nanoparticles as versatile therapeutic agents. The synergy between bacteriocins and nanometal oxides not only enhances antibacterial efficacy and disrupts virulence but also paves the way for novel interventions against infectious diseases and cancer.

The biological activity of nanoparticles is critically influenced by their morphology. Our TEM analysis revealed distinct shapes: spherical CuONPs, rod-like ZnONPs, and irregular, aggregated CuO–Zn composites. We propose that these morphologies directly underlie their mechanisms of action. The spherical CuONPs likely exhibit stronger antibacterial activity due to their high surface-to-volume ratio, which maximizes ROS generation and membrane interaction. The rod-shaped ZnONPs may instead facilitate physical penetration of cell walls. The irregular composite nanoparticles, with their heterogeneous surfaces, potentially enable synergistic effects through enhanced binding and sustained ion release. This structure-activity correlation has been added to the discussion to provide a stronger mechanistic interpretation of our results.

Copper oxide nanoparticles (CuO NPs) possess unique physicochemical properties, such as a high surface-to-volume ratio and reactivity, that make them attractive for a wide range of applications, including biomedicine, catalysis, and electronics⁴⁵.

The antimicrobial efficacy of CuO NPs operates through several synergistic mechanisms. They disrupt essential cellular processes and, similar to the role of copper in the innate immune system, catalyze the generation of bactericidal reactive oxygen species (ROS)⁴⁶. Compared to bulk copper, the nanoscale form provides a larger interactive surface and a higher rate of ion release, intensifying these effects. This multi-target mode of action simultaneously disrupts multiple cellular functions, reducing the likelihood of bacteria developing resistance⁴⁷.

Supporting the findings of Lina and Kadhim (2023)⁴⁸, who showed that zinc and silver oxide nanoparticles suppress swarming-regulating genes (wosA and rsbA), our study confirms that nanoparticles can modulate bacterial virulence. The rsbA gene, which encodes a membrane-embedded histidine kinase critical for initiating and maintaining swarming motility⁵⁰, is a key target. Its downregulation by biologically synthesized silver nanoparticles has been previously demonstrated⁴⁹.

The mechanism behind this suppression likely involves the disruption of cellular signaling. For instance, nanoparticles such as AgNPs and ZnO-NPs are known to interfere with quorum sensing, thereby inhibiting the expression of swarming-related genes⁵¹. In our work, ZnO-NPs exhibited stronger suppression of rsbA than CuO-NPs. This enhanced efficacy can be attributed to ZnO-NPs’ superior membrane-damaging ability, their capacity to disrupt cell signaling pathways, and their advantageous physical properties—such as smaller size, larger surface area, and higher cellular bioavailability—which facilitate more effective penetration and inhibition of virulence factors^52,53.

Conclusion

Our findings confirm that green-synthesized copper oxide and zinc oxide nanoparticles effectively inhibit the swarming motility of Proteus mirabilis. This anti-swarming activity, validated through both phenotypic observation and gene expression analysis, was linked to the significant downregulation of the rsbA gene—a central regulator of swarming. The stronger inhibitory effect of ZnO-NPs on rsbA highlights their enhanced potential for virulence control. Consequently, this research underscores the promise of bacteriocin-based nanoparticle formulations as effective and sustainable alternatives for managing opportunistic bacterial infections.

Electronic Supplementary Material

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Author contributions

- **Mais Emad Ahmed: Conceptualization, methodology, investigation, writing—original draft preparation.** (Corresponding author: (mais.emad@sc.uobaghdad.edu.iq).- Waqar Adnan Hamdan Al Kubaisi: Data curation, methodology, writing—review and editing, supervision.- **Hiba Mahdi Mahmood Alhashimi: Formal analysis, validation, methodology, and writing—review and editing.**- **Mohammad Y. Alfaifi: Formal analysis, methodology, validation, and resources.**- **Ali A. Shati: Visualization, software, and statistical analysis.**- **Serag Eldin I. Elbehairi: Investigation and experimental support.**- **Mohammed Aufy: Validation, Writing—review and editing, resources.**- **Ahmed M. Hussein: Conceptualization, Project administration, Validation, funding acquisition, writing—review and editing** (*Corresponding author: ahmed.hussein@univie.ac.at*).

Funding

The authors extend their appreciation to the University Higher Education Fund for funding this research work under Research Support Program for Central labs at King Khalid University through the Project number (CL/CO/B/3).

Data availability

The authors confirm that all data supporting the findings of this study are included within the article and its supplementary materials. Sequencing datasets generated and analyzed during the current study are available in the NCBI repository under accession number NR_042439.1 (submission ID: SUB15311522). Lactobacillus helveticus strains are available from the American Type Culture Collection (ATCC) under several strain numbers, including ATCC 15009 (commonly used in research; DSM 20075) and ATCC 10797. Additional strains include ATCC 7995, ATCC 12046, and ATCC 27558. Further genomic information can be accessed through the ATCC Genome Portal.https://www.atcc.org [https://share.google/ifMdDY5pbN9 × 6FIWx] [https://share.google/Tupd1lKFENMh78sjf] ATCC Genome Portal [https://share.google/688tYToeCf480rX3u]. All datasets generated in this study have also been deposited and are publicly available at the following repository: https://doi.org/10.5281/zenodo.19452878.

Declarations

Competing interests

The authors declare no competing interests.