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Inhibitory effects of nafcillin and diosmin on biofilm formation by Salmonella Typhimurium

BMC Microbiology volume 24, Article number: 522 (2024) Cite this article

Abstract

Objective

The foodborne pathogen Salmonella enterica serovar Typhimurium causes self-limiting gastroenteritis in humans and is difficult to eliminate due to its ability to adhere to surfaces and form biofilms that exhibit high resistance to antimicrobial agents. To explore alternative strategies for biofilm treatment, it is essential to investigate novel agents that inhibit Salmonella biofilms.

Method

In this study, we investigated the minimum biofilm inhibitory concentrations (MBICs) and minimum biofilm eradication concentrations (MBECs) of nafcillin and diosmin, both previously identified as Lon protease inhibitors, against biofilms formed by S. Typhimurium. Furthermore, we examined the expression of genes associated with the type II toxin-antitoxin system to enhance our understanding of the impact of these inhibitors.

Results

The findings indicated a strong antibiofilm effect of nafcillin, with MBIC and MBEC values of 8 µg/mL and 32 µg/mL, respectively. These results were confirmed by field emission scanning electron microscopy (FE-SEM), which showed that biofilm formation was reduced in the presence of nafcillin. Additionally, it revealed morphological changes in the bacteria within the nafcillin-treated biofilms. Furthermore, gene expression analyses demonstrated a significant reduction in the expression of type II TA system genes following treatment with nafcillin and diosmin.

Conclusion

This study highlights the effectiveness of nafcillin in disrupting the biofilms of S. Typhimurium. These results suggest promising avenues for the development of novel therapeutic strategies targeting biofilms associated with S. Typhimurium.

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Introduction

Salmonella is a genus of rod-shaped, Gram-negative bacteria that poses a significant public health risk worldwide, resulting in notable morbidity and economic impacts [1]. This genus encompasses over 2,500 distinct serotypes, with Salmonella Typhimurium being one of the most commonly confirmed serotypes in the United States [2, 3]. Raw and undercooked poultry and meat products are major sources of Salmonella infections [4]. The rise in salmonellosis cases can be partly attributed to the presence of Salmonella biofilms on various food-contact surfaces in poultry and meat processing facilities [5].

Bacterial biofilms are communities of microorganisms in which the cells are frequently encased in a self-produced matrix of extracellular polymeric substances [6], facilitating their adherence to surfaces [7]. According to the National Institutes of Health, 65% of all microbial infections and 80% of chronic infections are associated with biofilm formation [8]. Bacteria within biofilms demonstrate increased resistance to antimicrobial agents and host immune responses [9], a phenomenon linked to the protective physical barrier of the extracellular matrix, metabolic dormancy, and persistence mechanisms [10]. The establishment of bacterial biofilms contributes to the resilience of these organisms and complicates the treatment of infections, posing significant challenges for traditional antimicrobial approaches [11]. Despite the difficulties in controlling and managing biofilm formation, targeting microbial biofilms is increasingly recognized as a relevant and promising therapeutic strategy [12].

Toxin-antitoxin (TA) systems are components of the bacterial epigenetic regulatory framework, playing a significant role in bacterial survival [13]. These TA modules typically consist of a stable toxin and an unstable antitoxin, often encoded within a single operon [14, 15]. The antitoxin, which can be either a protein or a small RNA molecule, neutralizes the effects of the toxin. Under stress conditions, proteases such as Lon can degrade antitoxin proteins, resulting in the release of toxins and activation of the TA system [16]. Generally, antitoxin genes are located upstream of their corresponding toxin genes, and transcription of the TA operon is self-regulated through the binding of the antitoxin or the toxin-antitoxin complex to the promoter region [17]. The degradation of the antitoxin alleviates transcriptional repression, leading to increased production of both antitoxin and toxin transcripts [18].

Recent research has broadened the understanding of TA modules beyond their traditional roles in regulating genetic material to include a range of biological functions [19]. These functions encompass growth regulation, defense against phages, persistence, programmed cell death (PCD), general stress responses, and biofilm formation [19]. One of the earliest identified TA systems linked to biofilm formation is the MqsR system in Escherichia coli, which functions as a type II TA system alongside MqsA [20]. Moreover, comparative transcriptomic and proteomic analyses have demonstrated differential regulation of various TA systems between planktonic and biofilm cells [21]. For example, multiple TA systems have been found to be upregulated in biofilm cells of Treponema denticola, an oral spirochete associated with chronic periodontitis [22].

Additionally, the Lon protease plays a vital role in bacterial physiology, influencing motility, biofilm formation, and virulence across numerous bacterial species [23]. Comprising six identical subunits arranged in a hexameric ring characteristic of AAA + ATPase domains, Lon regulates bacterial metabolism and activates type II TA systems by degrading antitoxin proteins [24]. In a previous study, we explored drug repositioning strategies aimed at targeting the Lon protease in S. Typhimurium, identifying diosmin and nafcillin as effective inhibitors of Lon protease activity [25]. This study investigates the potential impact of diosmin and nafcillin as Lon protease inhibitors on S. Typhimurium biofilm formation, particularly considering the role of type II TA systems in this process.

Materials and methods

Bacterial strains and growth condition

In this study, we utilized S. Typhimurium ATCC 14028 alongside ten clinical isolates obtained from the Microbiology Department at Iran University of Medical Sciences. The invA and mdh genes were used to reconfirm the S. Typhimurium serotype using a PCR test [26, 27]. These isolates were conserved at − 80 °C in Brain Heart Infusion (BHI) broth supplemented with 20% glycerol.

Antimicrobial resistance pattern

Antibiotic resistance patterns of clinical isolates were investigated using disk diffusion methods according to the Clinical and Laboratory Standards Institute’s instructions [28]. The antimicrobial agents (MAST Co., UK) used in this study were as follows: tetracycline (30 µg), trimethoprim/sulfadiazine (1.25/23.75 µg), ciprofloxacin (5 µg), ampicillin (10 µg), chloramphenicol (30 µg), nalidixic acid (30 µg), cefotaxime (30 µg), ceftriaxone (30 µg), imipenem (10 µg), and azithromycin (15 µg). The E. coli ATCC 25922 strain was used for quality control purposes.

Biofilm assay

The biofilm assay for S. Typhimurium ATCC 14028 and, ten clinical isolates was conducted using a microtiter plate (MTP) method with 96-well flat-bottom polystyrene plates (Maxwell), following the protocols established by Wang some modifications [29].

Overnight cultures of S. Typhimurium isolates were diluted to 108 CFU/mL in Luria-Bertani (LB) medium without sodium chloride. A volume of 200 µL was added to each well of the microtiter plates, and the plates were incubated for 24 h at 30 °C under static conditions. After incubation, the medium was carefully removed, and the wells were washed three times with phosphate-buffered saline (PBS) before allowing them to air-dry for 20 min. The attached bacteria were fixed using 200 µL of methanol for 20 min; afterward, the plates were emptied and left to dry.

To stain the biofilms, 1% crystal violet was applied for 15 min, followed by two washes with distilled water to eliminate excess stain. The plates were then allowed to air dry for 30 min. Residual crystal violet was solubilized with 200 µL of 95% ethanol per well, and optical density (OD) at 570 nm was measured using an ELISA reader (Thermo Fisher Scientific, USA). LB medium without sodium chloride served as a negative control in all biofilm assays. Each experiment was conducted independently three times at varying time points.

The optical density cut-off (ODc) was defined as three standard deviations above the mean OD of the negative control. Based on their adherence capabilities, all isolates were classified into four categories: non-biofilm formers (OD ≤ ODc), weak biofilm formers (ODc < OD ≤ 2 × ODc), moderate biofilm formers (2 × ODc < OD ≤ 4 × ODc), and strong biofilm formers (OD > 4 × ODc) [30].

Determination of minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC)

The Minimum Biofilm Inhibitory Concentration (MBIC) for each compound was assessed by allowing bacteria to form biofilms on 96-well polystyrene microtiter plates in LB broth without sodium chloride, incorporating various concentrations of nafcillin and diosmin for 24 h at 30 °C.

After the incubation period, as described above, the plate was removed from the incubator, washed with PBS, fixed with methanol, and then stained with crystal violet. The dye was dissolved in ethanol, and the optical density (OD) of the wells was measured at 570 nm using an ELISA reader. Pure sterile growth medium (LB broth without sodium chloride) served as a negative control. The MBIC for nafcillin and diosmin was defined as the lowest concentration that completely prevented biofilm formation, indicated by biomass levels not significantly differing from those of the negative control [31]. A well containing LB medium and bacteria without any compounds served as the positive control. Each experiment was conducted in triplicate.

To determine the Minimum Biofilm Eradication Concentration (MBEC), planktonic suspensions were carefully removed following biofilm formation, and each well was washed twice with PBS. Subsequently, 200 µL of LB broth without sodium chloride was added to each well, containing various concentrations of nafcillin and diosmin, followed by incubation at 30 °C for 24 h. After incubation, survivors were enumerated for each treatment through serial dilution and plate counting on TSA, following removal from surfaces by gentle scraping with a plastic pipette tip and subsequent incubation at 37 °C for 24 h. For each bacterial strain, the MBEC was defined as the lowest concentration that resulted in at least a six-log reduction in biofilm population compared to the control (the well containing LB medium and bacteria without compounds) [32]. Each experimental setup was repeated three times.

RNA extraction, cDNA synthesis, and real-time PCR (RT–PCR) analysis

To investigate the impact of diosmin and nafcillin on the expression of lon protease, csgD, and type II TA systems in biofilm formation, total RNA was isolated from planktonic cells, as well as biofilm structures of S. Typhimurium isolates, and S. Typhimurium isolates that formed biofilms in media containing nafcillin (4 µg/mL) and diosmin (2 mg/mL).

For the planktonic cells, bacterial colonies from the two isolates (ATCC and clinical) were cultivated overnight at 37 °C in 5 mL of LB broth. The overnight culture was diluted to 108 CFU/mL in LB medium, and after 24 h of incubation, RNA extraction was performed on this culture. For biofilm samples, wells were washed twice with sterile PBS, and cells were collected via sonication and scraping in 200 µL PBS.

RNA extraction from the samples was conducted using the TRIzol reagent method [33]. The yield and purity of RNA were assessed using a spectrophotometer at 260/280 nm and 260/230 nm (NanoDrop, Thermo Fisher Scientific, USA). Subsequently, DNase1 treatment was applied as per manufacturer guidelines (Thermo Scientific, USA). RNA was then reverse-transcribed using the AddScript cDNA synthesis kit (AddBio, South Korea) according to the manufacturer’s instructions. The primer sequences utilized in this study are provided in Table 1.

Table 1 Primers used for qRT-PCR
Full size table

Quantitative reverse transcription-PCR (qRT-PCR) was performed with three technical replicates for each sample using a Rotor-Gene thermal cycler (Qiagen, Germany) employing the SYBR Green method (Ampliqon Co, Denmark). The total reaction volume (20 µL) included 1 µL of cDNA, 10 µL of SYBR Green master mix, 7 µL of nuclease-free water, and 1 µL of each primer. The thermal cycling protocol consisted of an initial denaturation step at 95 °C for 12 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 25 s. The invA gene served as the reference gene for normalizing gene expression levels. Relative fold changes in expression levels were calculated using the delta-delta Ct method [34].

Light microscopy analysis

To visualize the effects of nafcillin and diosmin on biofilm formation by S. Typhimurium ATCC 14028 and one clinical isolate, biofilms were established on glass cover slips placed in each well of a 6-well plate. The cover slips were inoculated with a bacterial suspension (108 CFU/mL in LB medium) treated with 4–8 µg/mL nafcillin and 1 or 2 mg/mL diosmin.

Following incubation at 30 °C for 24 h, the fluid in each well was discarded, and the plates were washed twice with PBS. The coverslips were then removed and stained with 1% crystal violet for 20 min. After staining, the coverslips were washed three times with 200 µL of distilled water to eliminate excess dye and air-dried. A light microscope (BX53, Olympus, Tokyo, Japan) was employed to observe the stained biofilms.

Field-emission scanning electron microscopy (FESEM)

The impact of nafcillin and diosmin on bacterial biofilm formation was investigated using Field-emission scanning electron microscopy (FESEM) following a previously established protocol with minor adjustments [35].

Suspensions of S. Typhimurium ATCC 14028 and one clinical isolate (at a concentration of 108 CFU/mL) were prepared in LB broth supplemented with 4 and 8 µg/mL nafcillin and 1 and 2 mg/mL diosmin. The samples were incubated for 24 h without shaking at 30 °C on glass coverslips within a 6-well polystyrene plate to facilitate biofilm formation. Following incubation, the fluid in each well was discarded, and the plates were washed twice with sterile water. Then, the samples were fixed for 4 h using 2.5% glutaraldehyde, rinsed three times with distilled water, and post-fixed with 1.5% osmium tetroxide for 1 h. After washing the coverslips twice with distilled water, the cells were dehydrated using a sequential ethanol series (30–100%) for fixation. The coverslips were then air-dried at room temperature and coated with gold before examination under a field-emission scanning electron microscope (MIRA3, TESCAN Co., Czechia).

Statistical analysis

Statistical analyses were conducted using GraphPad Prism 8 (GraphPad Software, Inc.). A one-way analysis of variance (ANOVA) was employed, followed by Tukey’s range test, with significance set at p < 0.05.

Results

Antibiotic sensitivity tests

Among the 10 isolates of S. Typhimurium, 7 (70%) were resistant to nalidixic acid, and 2 (20%) showed resistance to tetracycline; 2 (20%) were resistant to sulfamethoxazole/trimethoprim; 1 (10%) was resistant to chloramphenicol; and 1 (10%) was resistant to ampicillin. However, all of the S. Typhimurium isolates were susceptible to ciprofloxacin, cefotaxime, ceftriaxone, azithromycin, and imipenem.

Biofilm formation ability

The biofilm formation capacity of S. Typhimurium ATCC 14028 and ten clinical isolates was assessed using a microtiter plate (MTP) method. The results indicated that, based on the optical density (ODc), five isolates demonstrated weak biofilm formation, four exhibited moderate biofilm production, while S. Typhimurium ATCC 14028 and one clinical isolate formed strong biofilms (Fig. 1). These isolates were selected for further testing.

Fig. 1
figure 1

Biofilm formation ability (OD570) of S. Typhimurium isolates obtained by the Microtiter plate assay. OD cut-off (ODc) is a dashed line, which is applied to distinguish between non, weak, moderate, and strong biofilm former isolates

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Determination of MBIC and MBEC for each compound

The antibiofilm activity of nafcillin and diosmin on biofilm formation is illustrated in Fig. 2. The minimum biofilm inhibitory concentration (MBIC) for nafcillin was found to be 8 µg/mL, which effectively inhibited biofilm formation in both tested isolates (p < 0.0001). In contrast, diosmin did not achieve complete inhibition at any tested concentration; however, at a concentration of 2 mg/mL, it resulted in a significant reduction in biofilm formation compared to the positive control. Specifically, a decrease of 52% (p = 0.002) was observed in the ATCC isolate, while the clinical isolate showed a 64% (P = 0.001) reduction in biofilm volume.

Fig. 2
figure 2

Biofilm formation (A570 nm) of S. Typhimurium ATCC and clinical isolate in the presence of different concentrations of Nafcillin and Diosmin

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The minimum biofilm eradication concentration (MBEC) values are presented in Fig. 3. The results indicated that a concentration of 32 µg/mL of nafcillin led to a reduction of more than 6 logs (p < 0.0001) in both isolates, establishing it as the MBEC for this agent. For diosmin, although none of the concentrations examined achieved a reduction of more than 6 logs in the biofilm structure, a significant decrease of 1.2 logs (p = 0.019) was observed in the ATCC isolate, and a reduction of 1.86 logs (p < 0.0001) was noted in the clinical sample at a concentration of 0.5 mg/mL of this compound.

Fig. 3
figure 3

Log reductions of S. Typhimurium ATCC and clinical isolate in the presence of three different concentrations of Nafcillin and Diosmin

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Relative gene expression

Based on their capacity to form robust biofilms, two isolates) S. Typhimurium ATCC 14028 and a clinical isolate (were selected for analysis of relative gene expression in biofilm versus planktonic cells. Our RT-qPCR data revealed that all examined genes in these isolates exhibited higher expression levels compared to the control sample (Fig. 4). Notably, the relE/relB genes demonstrated the most significant increase in expression within the biofilm structures of both isolates.

Fig. 4
figure 4

Analysis of relative expression level of type II TA system genes in the presence of Nafcillin and Diosmin in S. Typhimurium ATCC and clinical isolate. Each group of genes is represented by three bars: the first bar representing the expression of genes in biofilm, the second bar representing the biofilm in the presence of Nafcillin (4 µg/mL), and the third bar representing the biofilm in the presence of Diosmin (2 mg/mL). Normalization of relative expression was performed using the reference gene invA, and error bars denote the standard deviations calculated from three biological replicates (P < 0.0001 using one-way ANOVA)

Full size image

To assess the impact of specific compounds on gene expression, biofilm formation was induced in media containing 4 µg/mL nafcillin and 2 mg/mL diosmin. Gene expression was then evaluated relative to a control group (biofilm without the addition of drugs). The findings indicated that both diosmin and nafcillin led to a reduction in gene expression in both isolates compared to the control.

Light microscopy and FESEM observations

The biofilm of S. Typhimurium isolates was examined using light microscopy at ×400 magnification (Fig. 5). It was observed that biofilm formation was significantly diminished in the presence of 4 µg/mL nafcillin on the coverslips, whereas a concentration of 8 µg/mL nafcillin completely inhibited biofilm development.

Fig. 5
figure 5

Inhibitory effect of Nafcillin and Diosmin on the biofilm formation of S. Typhimurium assessed by light microscopy (at 400x magnification)

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Figure 6 shows FESEM observations at ×10,000 magnification, revealing morphological changes in bacteria within nafcillin-treated biofilms; cells exhibited elongated and irregular shapes, accompanied by a reduction in the exopolysaccharide matrix. In contrast, biofilms treated with diosmin did not display significant changes in bacterial morphology, but there was a notable decrease in both bacterial numbers and the exopolysaccharide matrix.

Fig. 6
figure 6

Inhibitory effect of Nafcillin and Diosmin on the biofilm formation of S. Typhimurium assessed by scanning electron microscopy at × 10,000 magnifications

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Discussion

S. Typhimurium is a prominent pathogen associated with foodborne illnesses, frequently forming biofilms that enhance its resistance to antibiotics and environmental stressors [1, 36]. The biofilm formation presents significant challenges in both clinical and food safety contexts [37]. Given the critical role of biofilms, recent research has explored various strategies to mitigate their formation, including the use of antibiotics and natural compounds [32, 38]. This study focuses on nafcillin, a penicillinase-resistant beta-lactam antibiotic [39], and diosmin, a flavonoid with known anti-inflammatory properties [40] and identified as a Lon protease inhibitor in prior research [25], to evaluate their potential effects on biofilm formation.

S. Typhimurium utilizes diverse signaling and genetic pathways to initiate and sustain biofilm development, making it essential to identify effective agents for disrupting this process [41]. Literature indicates that type II toxin-antitoxin (TA) systems and proteolytic activity, particularly involving Lon protease, are critical factors influencing biofilm formation [42]. Type II TA systems consist of a stable toxin and a labile antitoxin. When the antitoxin is degraded by proteases such as Lon protease, the toxin can act, typically resulting in growth inhibition and dormancy [14]. Recent studies have illuminated the contributions of TA systems to biofilm formation; for example, the RelBE system has been implicated in biofilm formation in Vibrio cholerae [43], while a five-TA system deletion in E. coli led to increased expression of YjgK, which influences biofilm formation and reduces biofilm development at 8 h [44]. Additionally, the ParE toxin in E. coli triggers the SOS response, inhibits cell division, and promotes biofilm formation [45]. In Streptococcus pneumoniae, the YefM-YoeB system has been associated with oxidative stress and biofilm formation [46]. In our study, we examined the relative expression of five TA system genes in two robust biofilm-forming isolates of S. Typhimurium. Our results demonstrated increased expression levels across all genes investigated. Notably, the relEB genes in both isolates showed significant upregulation, with a 25.6-fold increase for relE and a 34.5-fold increase for relB in the ATCC strain, and a 23.56-fold increase for relE and a 31.01-fold increase for relB in the clinical isolate, compared to the control group. A related study highlighted that relEB2 and mazEF exhibited the highest expression levels among biofilm-associated TA systems in Klebsiella pneumoniae [47], further emphasizing the role of these genes in biofilm formation.

Lon protease is an ATP-dependent protease crucial for protein quality control and the regulation of various cellular processes, including biofilm formation [48]. Research indicates that inhibiting Lon protease can significantly reduce biofilm formation across several bacterial species [48]. Notably, in Pseudomonas aeruginosa, Lon protease is induced by subinhibitory concentrations of aminoglycosides and is essential for both biofilm formation and motility [49]. Additionally, Lon protease is involved in the degradation of regulatory proteins, such as antitoxins, which modulate gene expression related to biofilm development [50]. Moreover, Lon protease regulates the expression of genes implicated in curli fimbriae and cellulose production, including the CsgD transcription regulator, thereby facilitating biofilm establishment and maturation [51]. In our study, we observed a significant increase in the expression of the csgD gene in the biofilm structure of S. Typhimurium, with a 15.42-fold increase in the ATCC isolate and a 9.5-fold increase in the clinical isolate. Notably, the expression of this gene, along with all other studied genes, was diminished in biofilms formed in the presence of nafcillin or diosmin, both of which are known inhibitors of Lon protease, as indicated by previous research [25].

Nafcillin primarily targets cell wall synthesis in Gram-positive bacteria, where its beta-lactam structure interferes with peptidoglycan synthesis [52]. Research indicates that nafcillin can reduce biofilm biomass and has the potential to synergize with other agents, thereby enhancing overall anti-biofilm activity [53, 54]. Park et al. investigated the effects of sub-minimum inhibitory concentrations (MIC) of nafcillin and other antibiotics (1/256–1/2 × MICs) on Meticillin-resistant Staphylococcus aureus (MRSA) biofilm formation [55]. Their results indicated that sub-MICs of nafcillin increased biofilm formation, while the 1/2 × MIC inhibited biofilm formation in Staphylococcus aureus. In contrast, our study found that none of the concentrations examined led to an increase in biofilm formation. These findings highlight the differing effects of antibiotics on biofilm formation in various bacterial species, potentially due to differences in drug targets. The pronounced inhibitory effect of nafcillin on S. Typhimurium may be attributed to its inhibition of Lon protease activity in this bacterium. Diosmin, a naturally occurring flavonoid glycoside predominantly found in citrus fruits, is recognized for its anti-inflammatory and antioxidant properties [40]. Recent studies have also highlighted its potential as an antimicrobial and anti-biofilm agent [56]. In our study, we determined the Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC) of nafcillin and diosmin against biofilms of S. Typhimurium. The MBIC of nafcillin was found to be 8 µg/mL, while the MBEC was 32 µg/mL. These results demonstrate nafcillin’s efficacy in inhibiting biofilm formation at low concentrations, indicating a high potential for anti-biofilm activity. In contrast, none of the diosmin concentrations tested were able to completely inhibit or eradicate the biofilm. However, at a concentration of 2 mg/mL, we observed a significant reduction in biofilm formation. Also, in our findings, diosmin demonstrated comparable efficacy to nafcillin in inhibiting TA system genes; however, its ability to effectively disrupt biofilm formation was lower than that of nafcillin. Biofilms are complex communities of microorganisms that exhibit increased resistance to antibiotics and host immune responses [57]. This resistance can be attributed to the protective extracellular matrix surrounding the biofilm [58], which may hinder the penetration of diosmin. These results highlight important considerations regarding the multifactorial nature of biofilm formation, suggesting that Lon protease inhibition may not fully eradicate biofilms. Additionally, the established antibiotic properties of nafcillin may enhance its ability to disrupt biofilms, resulting in alterations to bacterial structure. As shown in the imaging results, field emission scanning electron microscopy (FESEM) revealed morphological changes in nafcillin-treated bacteria within the biofilm, displaying elongated and irregular cell shapes.

In previous work, we established the Minimum Inhibitory Concentration (MIC) of nafcillin for S. Typhimurium as 16 µg/mL [25], with an MBIC for biofilms at 8 µg/mL. This suggests that nafcillin can effectively disrupt biofilm development at concentrations lower than those required to inhibit planktonic cell growth. This discrepancy could indicate that biofilm-associated bacteria exhibit different sensitivity profiles compared to their planktonic counterparts. The decrease in MBIC relative to MIC suggests that nafcillin helps prevent S. Typhimurium biofilm formation not only through its antibiotic properties but also by effectively inhibiting Lon protease.

Hendri et al. [59]. conducted a study on the morphology of E. coli bacteria in the presence of antibiotics. Their findings revealed that, in contrast to unexposed cells, E. coli subjected to selective antibiotics like amoxicillin-clavulanate and ceftriaxone experienced notable morphological changes, including elongation of the bacterial cell wall. This elongation is likely due to a strong interaction between the negatively charged bacterial membrane and the positively charged β-lactam antibiotics, which possess a high hydrophobic content [60]. In our study, nafcillin was found to be more effective than diosmin in inhibiting biofilm formation. Both light microscopy and field emission scanning electron microscopy (FESEM) confirmed this inhibition. Furthermore, FESEM imaging revealed morphological changes in bacteria within the treated biofilm, showing elongated cell shapes.

Conclusion

Our findings underscore the significant effects of nafcillin and diosmin against S. Typhimurium biofilms. Notably, nafcillin exhibited a much stronger capacity to inhibit and disrupt biofilm formation. Given the challenges associated with treating biofilm-related infections, the exploration of novel and effective agents may enhance patient outcomes and improve food processing strategies. Our results suggest that these agents represent a promising new approach for addressing the biofilm-related challenges posed by S. Typhimurium.

Data availability

No datasets were generated or analyzed during the current study.

Abbreviations

S. Typhimurium:

Salmonella Typhimurium

MBIC:

Minimum Biofilm Inhibitory Concentrations

MBEC:

Minimum Biofilm Eradication Concentrations

MIC:

Minimum Inhibitory Concentrations

FE-SEM:

Field Emission Scanning Electron Microscopy

TA:

Toxin-Antitoxin

MTP:

Microtiter Plate

CFU:

Colony Forming Unit

PBS:

Phosphate-Buffered Saline

OD:

Optical Density

RT–PCR:

Real-Time PCR

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Acknowledgements

This study was financially supported for a PhD thesis in Iran University of Medical Sciences (Tehran, Iran), for which we are very grateful.

Funding

This study was financially supported by a research grant (No.24791) in Iran University of Medical Sciences (Tehran, Iran).

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Authors and Affiliations

  1. Microbial Biotechnology Research Center, Iran University of Medical Sciences, Tehran, Iran

    Negar Narimisa, Amin Khoshbayan, Shabnam Razavi & Faramarz Masjedian Jazi

  2. Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

    Negar Narimisa, Amin Khoshbayan, Shabnam Razavi & Faramarz Masjedian Jazi

  3. Laboratory of Bioinformatics and Drug Design (LBD), Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

    Sajjad Gharaghani

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  1. Negar Narimisa
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  2. Amin Khoshbayan
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Contributions

Conceptualization: N. N.Methodology: N. N., and S. G.Data curation: N. N., and, A. K.Writing- Original draft preparation: N. N.Visualization and Investigation: Sh. R., and F. M. J.Supervision: Sh. R., and F. M. J.Writing- Reviewing and Editing: All authors have read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Shabnam Razavi or Faramarz Masjedian Jazi.

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Ethical approval

This study was approved by the Ethics Committee of Iran University of Medical Sciences by Ethics No. (IR.IUMS.FMD.REC.1402.262).

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The authors declare no competing interests.

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Narimisa, N., Khoshbayan, A., Gharaghani, S. et al. Inhibitory effects of nafcillin and diosmin on biofilm formation by Salmonella Typhimurium. BMC Microbiol 24, 522 (2024). https://doi.org/10.1186/s12866-024-03646-1

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Keywords

BMC Microbiology

ISSN: 1471-2180

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