Antimicrobial and antibiofilm activities of supernatants of Lactiplantibacillus plantarum A2 and Lactiplantibacillus plantarum 2.1 against Escherichia coli ATCC 25922

doi:10.21203/rs.3.rs-4978949/v1

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Antimicrobial and antibiofilm activities of supernatants of Lactiplantibacillus plantarum A2 and Lactiplantibacillus plantarum 2.1 against Escherichia coli ATCC 25922

https://doi.org/10.21203/rs.3.rs-4978949/v1

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Background

Urinary tract infections are the most common infections in humans. Furthermore, they are relevant for public health for being closely related to the phenomenon of dispersion of multi-resistant strains of bacteria. Most cases are caused by Escherichia coli, a commensal microorganism that colonizes the urinary tract by translocating from the gastrointestinal tract. In this context, the present work was dedicated to finding alternatives for controlling the dispersion of its multidrug-resistant strains by studying the inhibitory effect of the cell-free supernatants of Lactiplantibacillus plantarum A2 (LPA2) and Lactiplantibacillus Plantarum 2.1 (LP2.1). Among a group of eight strains isolated from cocoa fermentation, these two stood out in terms of their probiotic potential and possible applications in the health sector. This study conducted the in vitro investigation of the antimicrobial and antibiofilm activities of both supernatants and the preliminary analysis of their composition.

Results

Both supernatants evaluated showed antimicrobial and antibiofilm activity against E.coli ATCC 25922. In the semi-solid agar diffusion assay, LPA2 formed a 17 mm inhibition halo and LP2.1 formed a 12 mm one. In the 96-well microplate assay, LPA2 showed 89.22% inhibition and LP2.1 showed 91.22%. Regarding antibiofilm activity, LPA2 showed 80.96% inhibition and LP2.1 showed 81%. Preliminary analysis of the metabolites indicated that thermostable substances dependent on acidic pH may be responsible for the anti-E.coli action observed in the experiments.

Conclusions

LPA2 and LP2.1 supernatants are capable of antagonizing E.coli ATCC 25922 in vitro and can be used to control its proliferation in cases of asymptomatic bacteriuria, maintain the balance of the microbiota, and reestablish the microbiota after antimicrobial therapy.

lactobacilli

cell-free supernatants

antimicrobial activity

antibiofilm activity

bioactive molecules

Lactiplantibacillus plantarum are Gram-positive bacteria that belong to the phylum Firmicutes. Within this phylum, L.plantarum is included in the class Bacilli and, subsequently, in the order Lactobacillales (Cui et al., 2019; Zheng et al., 2020). L.plantarum presents a coccobacillus morphology and is considered a flexible habitat species, able to survive in different environments, such as the mammalian gastrointestinal tract, vaginal mucosa, food matrices, the ground, and niches associated with vegetables (Mani-López et al, 2022). It is most traditionally used in the food industry to improve the sensory aspects of food and increase its nutritional value (Echegaray et al., 2023).

However, L.plantarum also has properties that enable its use in healthcare, such as antimicrobial and antibiofilm activity, resistance to adverse gastrointestinal transit conditions, and ability to adhere to mucous membranes (Onbas et al., 2019; Jung et al., 2019; Wieers et al., 2020; Rocchetti et al., 2021). Regarding antimicrobial activity, it occurs through several mechanisms, such as competition for nutrients and adhesion sites, production of metabolites that can act on different microorganisms, and modulation of the immune system (Bajaj et al., 2015; Pereira et al., 2018; Fidanza et al, 2021). As for its anti-biofilm activity, recent publications have demonstrated that lactobacilli supernatants can inhibit biofilm formation (Kiousi et al., 2023) or disorganize the structure of already-formed biofilms (Carvalho et al., 2021). The substances responsible for this inhibitory effect are produced during the fermentative metabolism of L.plantarum and include organic acids, bacteriocins, exopolysaccharides, and biosurfactants, among others (Pereira et al., 2018).

In vitro studies have already demonstrated that the substances produced during the metabolism of L.plantarum - also called cell-free supernatants - are capable of inhibiting different types of microorganisms, such as Gram-positive bacteria (Arief et al., 2015; Yan et al., 2019; Lee et al., 2021; Shaaban et al., 2022), Gram-negative bacteria (Khodaii et al., 2017; Chen et al., 2019; Pazhoohan et al., 2020; Selis et al., 2021), and yeast (Srivastava et al., 2020; Pino et al., 2022). Furthermore, after ingestion, L.plantarum develops mechanisms of adaptation to the intestinal environment that involve interaction with resident microorganisms and with factors intrinsic and extrinsic to the host, such as age and diet (Huang et al., 2021). Adapting to the intestinal environment produces evolutionary forces capable of remodeling the composition of the intestinal microbiota and, consequently, modulating the physiological functions of the host (Echegaray et al., 2023). Studies focused on the inhibitory effect exerted by cell-free supernatants have become especially relevant after antimicrobial resistance was considered one of the three main health threats of the 21st century by the World Health Organization (Asenjo et al., 2021). Furthermore, the use of antimicrobials is associated with an imbalance of the endogenous microbiota, leading to episodes of diarrhea. In cases where infections are recurrent and, therefore, their treatment, these events become frequent and uncomfortable.

Urinary tract infections (UTIs) are the most common infections in humans, with 20% of their occurrence reported as recurrence (persistence or reappearance of the same bacteria) and 80% as reinfection, when caused by bacteria other than the one responsible for the first infection (Pigrau et al., 2020; Naziri et al., 2021). UTIs can be community or hospital-acquired and, in both cases, the microorganism predominantly isolated from them is Escherichia coli, followed by Klebsiella pneumoniae (Flores-Mireles et al, 2015; Zhao et al, 2020; Naziri et al, 2021). Normally, E.coli is considered a commensal member of the intestinal microbiota (Jang et al, 2017; Daga et al, 2019). However, depending on the virulence factors expressed, it can access the bloodstream, colonize other niches, and cause extraintestinal infections, such as UTIs, sepsis, and neonatal meningitis (Mora-Rillo et al., 2015; Daga et al., 2019). One of the most worrying virulence factors, from a public health point of view, is the ability to form biofilms since they are responsible for approximately 60% of all human infections (Preda et al., 2019).

Bacterial biofilms are made up of clusters of cells protected by exopolysaccharides produced by the same microorganisms that compose the biofilm (Jamal et al., 2018; Rather et al., 2021; Assefa et al., 2022). The biofilm formation process is sequential and involves four basic steps: adhesion of the microorganism to a surface; formation of microcolonies; maturation of microcolonies; and dispersion of cells to colonize new surfaces (Santos et al., 2018; Mirzaei et al., 2020). In addition to causing a considerable part of human infections, biofilm formation is also related to the dispersion of multidrug-resistant strains, as it allows bacterial cells to protect against the action of antimicrobials and escape the immune system, impairing the effectiveness of treatment and contributing to the dispersion of resistant strains (Shah et al., 2019; Zhao et al., 2020).

In this context, cell-free supernatants derived from the lactobacilli metabolism emerge as an alternative for controlling E.coli proliferation and maintaining the balance of the intestinal microbiota during and after antimicrobial therapy. Lactiplantibacillus plantarum A2 and Lactiplantibacillus plantarum 2.1 were analyzed by our group in a previous study and stood out in terms of probiotic potential among a group of eight strains of lactobacilli isolated from cocoa fermentation. In order to expand their application within the health sector, this study conducted an in vitro investigation regarding the antimicrobial and antibiofilm activities of their supernatants.

2.1 Conditions for growth and maintenance of microorganisms

The strains of both Lactiplantibacillus plantarum 2.1 and Lactiplantibacillus plantarum A2 were previously isolated, identified, and sequenced by our research group (Santos et al, 2016). These strains were stored in MRS plus 30% glycerol at -80ºC, and were cultivated in MRS (De Man, Rogosa and Sharpe, HiMedia), under microaerophilic conditions, at 37°C, for the assays. Escherichia coli ATCC 25922 was provided by the Mycology Laboratory of the State University of Santa Cruz and was maintained in Mueller-Hinton (KASVI) plus 30% glycerol at -80ºC.

2.2 Preparation of cell-free supernatants

To obtain cell-free supernatants, L.plantarum 2.1 and L.plantarum A2 were initially cultivated in MRS broth for 48 hours at 37ºC. After growth, the cultures were centrifuged at 8000 x g for 15 minutes. Then, the pellets were discarded, and the supernatants were collected. The recovered supernatants were filtered through 0.22 µm membranes and stored in a freezer at -20°C. Samples containing only MRS medium (no bacterial growth) were prepared. These samples were also filtered through 0.22 µm membranes before being stored in the freezer at -20ºC.

2.3 Antimicrobial activity

2.3.1 Semi-solid agar diffusion

For the semi-solid agar diffusion assay (Alameri et al., 2022), E.coli was inoculated in MRS broth for 18–24 hours at 37°C, under shaking. The following day, a suspension of E.coli at 10⁸ CFU/mL was prepared in MRS broth. Twenty µl of this suspension was inoculated into 7 mL of semi-solid MRS agar (0,7%). After solidification, wells were made in the agar with 200 µl tips and then these wells were filled with 100 µl of the lactobacilli supernatants (L.plantarum A2 and L.plantarum 2.1), and with 100 µl of the MRS control. Plates were incubated at 37°C for 18–24 hours. To verify the antimicrobial activity, the presence of the inhibition halo was observed and measured with a ruler.

2.3.2 96-well microplate assay

For the 96-well microplate assay (Scillato et al., 2021), E.coli was grown in Mueller-Hinton broth for 18–24 hours, under shaking. Then, a suspension of 10⁸ CFU/mL of E.coli was prepared and 100 µL of it was added to all the wells of a microplate. In the first ones, 100 µL of new and sterile Mueller-Hinton broth was added (wells without treatment). Then, 100 µL of LPA2, LP2.1, and MRS were added to the following wells separately. The microplate was incubated for 18–24 hours at 37°C. After incubation, the microplate was analyzed in an ELISA reader (VERSAmax Tunable, 600 nm). Then, the percentage of inhibition of the supernatants was calculated using the following formula: %Growth inhibition = 100x[1-(OD_T/OD_CN)], where OD_T refers to the optical density value of the wells containing lactobacilli and MRS supernatants and OD_CN refers to the well with untreated E.coli. The contents of the wells were plated on Mueller-Hinton agar plates with the aid of the replicator. Then, the plates were incubated at 37ºC for 18–24 hours and, after this, bacterial growth was observed.

2.4 Assessment of the biofilm formation capacity of Escherichia coli ATCC25922

The biofilm formation ability of E.coli ATCC 25922 was evaluated in a 96-well microplate, as described by Stepanovic et al. (2007) and Cepas et al. (2019), with some modifications. First, 200 µL of Luria-Bertani medium (LB, KASVI) plus 0.25% glucose (Exodus Científica) were added to all wells of the microplate. The wells intended for evaluating biofilm formation received 20 µL of E.coli suspension 10⁸ CFU/mL in LB plus 0.25% glucose. Then, the microplate was incubated at 37°C for 24 hours. The negative control for this assay was composed of LB plus 0.25% glucose, without bacterial growth.

After the incubation time, the samples were fixed with 200 µL of 96% methanol (Exodus Científica) for 20 minutes at room temperature. Next, the microplates remained on the bench in an inverted position, overnight. The following day, the microplates were stained with 150 µL of 2% crystal violet for 15 minutes at room temperature. After staining, 150 µL of 33% acetic acid (Êxodo Científica) was added to all wells, before analysis in an ELISA reader (595nm). Using the OD_595nm values found, the cutoff value of the microplate was obtained (OD_C = average OD_CN + 3 x standard deviation_CN) and from the cut-off value it was possible to obtain a final value of OD_595nm (OD_F = average OD_T - OD_C). These final OD_595nm values (OD_F) allowed us to classify E.coli into non-biofilm producer (OD_F ≤ OD_C), weak biofilm producer (OD_C < OD_F ≤ 2xOD_C), moderate biofilm producer (2xOD_C < OD_F ≤ 4xOD_C), or strong biofilm producer (4xOD_C < OD_F).

2.5 Inhibition of biofilm formation by lactobacilli supernatants

For this assay, 100 µL of LB medium, supplemented with sterile 0.25% glucose, was added to all wells of a 96-well microplate. The wells tested for biofilm inhibition received 100 µL of LPA2, LP2.1, and MRS without growth. Then, 20 µL of an E.coli bacterial suspension (10⁸CFU/mL) was added to them. The control well received 100 µL of LB medium, supplemented with 0.25% glucose and 20 µL of bacterial suspension at 10⁸ CFU/mL without treatment (Ghane et al, 2020). After preparation, the microplate was incubated at 37ºC for 24 hours. The steps for evaluating biofilm formation were performed: fixation with 96% methanol, staining with 2% crystal violet, and OD_595nm reading. The percentage of inhibition of biofilm formation was obtained according to the following formula: %Inhibition = 100 - [(OD₅₉₅ test x 100) / OD₅₉₅ control].

2.5 Biofilm visualization by scanning electron microscopy

Scanning electron microscopy was performed to observe the effect of supernatants on the ability of E.coli to adhere to the surface. Sample processing was conducted at the Electron Microscopy Center of the State University of Santa Cruz. For this, E.coli was inoculated in LB medium, supplemented with 0.25% glucose, and incubated at 37°C for 18–24 hours. The following day, coverslips were deposited into the wells of a 12-well microplate. From then on, the same protocol was followed to analyze the inhibition of biofilm formation. The microplate was incubated for 24 hours at 37°C. After this, the microplate was fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). The next step was dehydration in an increasing series of ethanol for 10 minutes each step: 30% alcohol, 50% alcohol, 70% alcohol, 90% alcohol, and 100% alcohol. After dehydration, the samples were dried in critical point equipment and the stubs were assembled. The images were obtained using a Quanta 250 scanning electron microscope (FEI Company 2010) at the SEM and TEM Microscopy Laboratory at the Federal University of Juiz de Fora.

2.6 Preliminary analysis of compounds present in supernatants

2.6.1 Evaluation of the influence of pH neutralization, heating to 100ºC and proteolysis on the inhibitory effect of supernatants

This assay analyzed the presence of organic acids, protein compounds and thermostable substances in the supernatants, using a 96-well microplate technique (Scillato et al, 2021). To evaluate the presence of these compounds, the supernatants needed to be previously treated as follows: for the presence of organic acids, the supernatants had their pH neutralized to 7.0 with a NaOH solution (1 molar); for the presence of thermostable substances, the supernatants were heated at 100°C for 15 minutes; and for the presence of protein compounds, supernatants were incubated with trypsin (Sigma - Aldrich) at a concentration of 2.5 mg/mL for 2 hours at 37°C (Selis et al, 2021).

To conduct this assay, E.coli was cultivated in Mueller-Hinton broth for 18–24 hours, under agitation. Then, a suspension of 10⁸ CFU/mL of E.coli was prepared and 100 µL of this suspension was added to all wells of a sterile microplate. In the first wells, 100 µL of sterile Mueller-Hinton broth was added (wells without treatment). The next wells received 100 µL of LPA2, LP2.1, and MRS without prior treatment. Finally, 100 µL of supernatants and previously treated MRS were added to another group of wells. The microplate was incubated for 18–24 hours at 37°C and, after incubation, the contents of the wells were plated on Mueller-Hinton agar plates with the aid of the replicator to check the antimicrobial activity.

2.6.2 Assessment of Biosurfactant Production

To evaluate biosurfactant production by lactobacilli, 2 mL of their supernatants were mixed with 2 mL of common kerosene (X9 Kerosene). The mixture was homogenized by vortexing for 2 minutes and then incubated at room temperature for 24 hours (Morais et al., 2017). The same procedure was performed for the MONTH without microbial growth, as an assay control. Based on the height of the mixture, the emulsifying activity index (E₂₄) could be calculated after 24 hours using the following formula: E₂₄ = emulsion layer height / total height of the mixture x 100 (Ciandrini et al., 2020).

2.7 Fourier Transform Infrared Spectroscopy (FTIR)

The Fourier transform infrared spectroscopy (FTIR), in attenuated total reflection (ATR) mode, was used to recognize the functional groups that make up the LPA2 and LP2.1 supernatants and evaluate the differences between them and MRS (Morais et al, 2017). For this, LPA2, LP2.1, and MRS were concentrated for 7 days in a speed vac (Concentrator 5301 - Eppendorf). The measurements were conducted in the spectral range between 650 and 4000 cm^− 1 using Spectrum 400 FT-IR/FT-NIR Spectrometer equipment from PerkinElmer. The spectra obtained were averages from 10 scans, with a resolution of 4 cm^− 1 and were processed using PerkinElmer's Spectrum software. Each supernatant and MRS sample generated a spectrum. These spectra were compared and the MRS spectrum was subtracted from the LPA2 and LP2.1 spectra, which allowed the spectra to be obtained referring only to the contents of the lactobacilli supernatants.

2.8 Statistical analysis

All assays were performed in triplicate. The values presented represent the mean ± standard deviation and were analyzed by GraphPrism 5.0. Statistical differences between values were determined using ANOVA and Tukey's post-test, with p˂0.05.

3.1 LPA2 and LP2.1 supernatants could inhibit E.coli growth

The two lactobacilli supernatants studied showed antibacterial activity, demonstrated through the semi-solid agar diffusion and the 96-well microplate assays. In the semi-solid agar diffusion assay, LPA2 formed an inhibition halo of 17 mm and LP2.1 formed an inhibition halo of 12 mm. The MRS control, in turn, did not form an inhibition halo (Fig. 1, in the Supplementary Material). In the microplate assay, lactobacilli supernatants also inhibited E.coli growth significantly compared to MRS. LP2.1 showed 91.22% inhibition and LPA2 showed 89.96%, while MRS showed only 48.15% (Fig. 1). Plating the contents of the microplate wells on Mueller-Hinton agar demonstrated the microbicidal profile of the action of the lactobacilli supernatants, since it was not possible to observe bacterial growth in the wells treated with these supernatants (Fig. 2, in the Supplementary Material).

3.2 E.coli ATCC 25922 is a moderate biofilm producer

In the present study, E.coli ATCC 25922 was classified as a moderate biofilm producer. This was possible due to a set of operations based on the OD_595nm values found during the analysis of the microplate in the ELISA reader. The calculated cutoff value for the microplate (OD_C = average OD_CN + 3 x standard deviation_CN) was equivalent to 0.21559 and the OD_F value (OD_F = average OD_T - OD_C) was equivalent to 0.58931. Replacing these values in the classification equation allowed us to categorize E.coli ATCC 25922 as a moderate biofilm producer (2xOD_C < OD_F ≤ 4xOD_C↔ 0.43118 < 0.58931 ≤ 0.86236).

3.3 LPA2 and LP2.1 supernatants inhibit biofilm formation by E.coli

After 24 hours of incubation, the lactobacilli supernatants were able to significantly inhibit the formation of the E.coli biofilm in relation to MRS. The inhibition percentages obtained were equivalent to 80.96% for LPA2, 81% for LP2.1, and 26.55% for the MRS control (Fig. 2). The biofilm structure, observed by scanning electron microscopy, demonstrated different degrees of integrity depending on the treatment received. The E.coli sample that did not receive treatment demonstrated a completely intact structure (Fig. 3a). The samples that were treated with LP2.1 (Fig. 3b) and LPA2 (Fig. 3c) did not present any biofilm structure, only a few isolated bacteria.

3.4 Preliminary analysis of compounds present in supernatants

3.4.1 pH neutralization suspends the inhibitory effect of supernatants

Without prior treatment, LPA2 and LP2.1 inhibited the growth of E.coli and MRS, conversely, they did not inhibit bacterial growth. Neutralizing the pH of both lactobacilli supernatants (LPA2 and LP2.1) to 7.0 suspended their inhibitory effect when compared to untreated supernatants. Differently, heating them at 100ºC for 15 minutes and treatment with trypsin (2.5 mg/mL) did not suspend their inhibitory effect (Table 1, Fig. 3 in Supplementary Material). The MRS sample without bacterial growth was subjected to the same treatments as the lactobacilli supernatants and it was possible to observe, as a result, that none of the three treatments significantly influenced its inhibitory effect against E.coli growth (Table 1, Fig. 3 in Supplementary Material).

Table 1

**Preliminary analysis of compounds in the supernatants in a 96-well microplate.** LPA2 and LP2.1 continued to inhibit *E.coli* even after heating to 100ºC and after treatment with trypsin. However, they lost the ability to inhibit *E.coli* after having their pH neutralized. MRS control did not inhibit *E.coli* under any of the conditions presented. (+) indicates that there was growth of *E.coli*.
Samples	Treatments
Samples	No treatment	Heating to 100ºC	Neutral pH	Trypsin
LP2.1	Inhibition	Inhibition	+	Inhibition
LPA2	Inhibition	Inhibition	+	Inhibition
MRS no growth	+	+	+	+

3.4.2 The emulsifying activity indexes of LPA2 and LP2.1 demonstrate the biosurfactant production of the respective strains

The emulsifying activity rates after 24 hours were equivalent to 76.67% for LPA2, and 73.33% for LP2.1, which were statistically different from those observed for MRS without bacterial growth (45%, Fig. 4).

3.5 Analysis of the functional groups that make up lactobacilli supernatants

The spectra of LPA2 and LP2.1 were analyzed in comparison to the MRS control, which enabled the analysis of the functional groups present in the supernatants as a result of the metabolism of L.plantarum A2 and L.plantarum 2.1 alone. Furthermore, obtaining these spectra also allowed the comparison between LPA2 and LP2.1. The infrared spectra of both supernatants were similar, with slight differences between them (Fig. 5), particularly in the range between 4000 − 1900 cm^− 1 (Fig. 5, Table 2), where LP2.1 presents the peak at 3620.71 cm^− 1. This peak indicates the presence of the hydroxyl functional group and was not observed in the LPA2 spectrum. LPA2 and LP2.1 also presented identical peaks, from 747.14 cm^− 1 to 1721.43 cm^− 1 (Table 2).

Table 2

**LPA2 and LP2.1 spectral bands in wavenumber (cm-1) and the possible connections they represent.** The table lists the common and distinct spectral bands between both supernatants studied.
Wavenumber (cm^− 1)	Possibles bonds
LP2.1
3620,71	O - H
2991,43	C - H
2519,29	O - H
1957,14	N - H; O - H
LPA2
2985,71	C - H
2525,71	O - H
1939,29	N - H; O - H
Wavenumbers commons
1721,43	C - H; C = O
1582,14	N - H
1455,00	CH₂
1231,43	C - O
1128,57	C - H secondary
1043,57	N - H; O - H; C - O
928,57	C - H
856,43	C - H
825,71	N - H
747,14	Methyl group

Urinary tract infections (UTIs) are the most common infections in clinical practice, especially in women of childbearing age (Foxman, 2014; Guglietta, 2017; Kenneally et al., 2022). From a public health perspective, UTIs represent an emerging concern due to their close relationship with the spread of antimicrobial-resistant strains. This relationship begins with the mistaken delimitation of asymptomatic bacteriuria and urinary tract infection. When there is an error in differentiation, asymptomatic bacteriuria is considered a UTI and the individual is unnecessarily subjected to the use of antimicrobials, favoring the development of resistant strains and recurrent urinary infections (Rossi et al., 2020; Lawati, Blair and Larnard, 2021). The recurrent use of antimicrobials in recurrent UTIs promotes a long-term change in the microbiome of the patient and contributes to the development of multidrug-resistant strains of bacteria (Kostakioti et al, 2012).

The core of this study was the bioactivity of LPA2 and LP2.1 supernatants against E.coli ATCC25922, mainly because this bacterium represents around 70% of the causative agents of UTIs, both in the community and hospital-acquired types (Grey et al., 2023). The usual mechanism by which E.coli causes urinary tract infections is the colonization of the urethra and bladder from the gastrointestinal tract, triggering an inflammatory response in the host (Riley, 2020; Lawati, Blair and Larnard, 2021). The development of the ability to adapt to a new nutritional niche, to grow and multiply in the urinary tract involves the expression of virulence factors related to biofilm formation by commensal strains of E.coli (Foxman et al., 2000; Shah et al., 2019). The most studied virulence factors so far are the adhesive organelles fimbria type 1 and fimbria “curli”, which act in the initial stage of biofilm formation and are responsible for bacteria-surface interaction and cell-cell communication, strengthening the maturation of the structured biofilm (Shah et al., 2019; Ruhal e Kataria, 2021). The biofilm organization also promotes the development of multidrug-resistant strains and their subsequent dispersion, as it delays the penetration of antimicrobial agents through the extracellular matrix (Soto et al., 2006; Dumaru et al., 2019).

Given the problems related to the increase in the dispersion of multi-resistant strains, as well as the financial burden on health systems, the National Health Surveillance Agency (ANVISA) banned the sale of antimicrobials without a medical prescription in Brazil in October 2010. However, this measure was not enough to reduce the occurrence of multidrug-resistant strains (Rodrigues et al., 2021). Therefore, this study analyzes the viability of LPA2 and LP2.1 supernatants as a tool for controlling the proliferation of E.coli in cases of asymptomatic bacteriuria in healthy, non-pregnant women, reducing the need for traditional antimicrobials, stopping the development of multi-resistant strains and preserving the balance of the microbiota. Therefore, we began the study with the evaluation of antimicrobial activity using the semi-solid agar diffusion assay and the 96-well microplate assay. In the semi-solid agar diffusion assay, LPA2 exhibited a 17 mm inhibition halo and LP2.1 showed a 12 mm one, whereas the MRS control did not form an inhibition halo (Fig. 1, in the Supplementary Material). The inhibition effect of the supernatants was repeated in the 96-well microplate assay, with LPA2 reaching 89.96% inhibition and LP2.1 reaching 91.22% (Fig. 1). Unlike in the semi-solid agar diffusion assay, MRS showed a slight inhibitory effect in the microplate assay, but it was not statistically significant when compared to those of the supernatants (Fig. 1).

Lactobacilli are already recognized for their ability to antagonize other microorganisms through the production and release of antimicrobial substances, which can act by inhibiting adhesion to surfaces, blocking their ability to invade tissues, and limiting their growth (Kim et al., 2019). The inhibition effect of L.plantarum supernatants, more specifically, has been demonstrated through various in vitro techniques over time. In semi-solid agar diffusion assays, the size of the inhibition zones against E.coli formed by L.plantarum supernatants generally varies between 10 mm and 20 mm, in studies evaluating antimicrobial activity (Shim et al., 2016; Chen et al., 2021; Fijan et al., 2022). In the techniques conducted in a 96-well microplate, it is possible to understand that the inhibitory effect of L.plantarum supernatants is only perceived in supernatants produced within 48 hours of strain growth. After 72 hours, their inhibitory effect is lost (Danilova et al., 2019). The choice to evaluate antimicrobial activity by two different methods considered the assumption that the method used can also affect the degree of the inhibitory effect, in addition to factors related to the probiotic strain and the bacteria strain targeted by the inhibition (Pazhoohan et al, 2020). Microplate tests are more sensitive to inhibition effects because they facilitate the diffusion of the supernatant (Scillato et al., 2021), which can be seen in the high percentages of inhibition obtained in the microplate assay (Fig. 1). In tests in petri dishes, the solid or semi-solid culture medium acts as a limiting factor for the diffusion of the supernatant. Thus, the fact that LPA2 and LP2.1 demonstrate inhibition capacity in both methods tested proves their potential for antimicrobial activity against E.coli.

In order to complement the information obtained on the inhibitory effect exerted by LPA2 and LP2.1, it was decided to also evaluate their capacity to inhibit the formation of E.coli biofilm. Firstly, it was necessary to classify E.coli according to its biofilm formation ability. This classification was based on the cutoff value (OD_C=0.21559) calculated for the microplate, which determines a threshold for biofilm formation of the studied strain (Stepanovic et al, 2007; Cepas et al, 2019; Naziri et al, 2021). From the cutoff value, the final OD value was calculated (OD_F = 0.58931), allowing the classification of E.coli as a moderate biofilm producer. After this, the ability of LPA and LP2.1 supernatants to inhibit biofilm formation was evaluated. Following 24 hours of treatment, LPA2 showed 80.96% inhibition, LP2.1 81%, and the MRS control 26.55% (Fig. 2). In general, studies evaluating antibiofilm activity demonstrate that lactobacilli supernatants of extraintestinal origin are efficient in inhibiting E.coli biofilm formation, with percentages ranging from 48.68–52.61% (Lee et al., 2020; Sornsenee et al., 2021). The percentages of inhibition obtained by the present study, combined with the electron microscopy findings (Fig. 3), indicate that the antibiofilm activity of LPA2 and LP2.1 occurs through membrane disruption and cell death during the initial phase of biofilm formation, since the untreated E.coli sample presented a completely intact biofilm structure (Fig. 3a), while those treated with LP2.1 (Fig. 3b) and LPA2 (Fig. 3c) did not present a formed biofilm structure. It is worth mentioning that images referring to the MRS control treatment were not obtained since its percentage of inhibition was not statistically significant when compared to those of LPA2 and LP2.1 (Fig. 2). In this study, MRS was used as a basis for the growth of lactobacilli and for the subsequent production of their supernatants. Therefore, it was used as a control for the bioactivity of LPA2 and LP2.1 throughout the work. The inhibitory effect of MRS is attributed to the presence of polysorbate 80 in its composition (Sloup et al., 2016; Wilson et al., 2021). As in the present study, the inhibition effect of MRS was not statistically significant when compared to that of LPA2 and LP2.1, it is possible to infer that the inhibitory effect demonstrated by LPA2 and LP2.1 is due to the production and release of biomolecules from the metabolism of the respective strains and not the presence of polysorbate 80.

The second part of our study aimed to understand - albeit indirectly - which metabolites produced by L.plantarum A2 and L.plantarum 2.1 are involved in the inhibitory effect against E.coli. For this purpose, three tests were performed: the antimicrobial activity test in microplates with previously treated and untreated supernatants, the biosurfactant production test, and, by FTIR, the analysis of functional groups. The results obtained from the microplate assay indicated the presence of thermostable and acid pH-dependent substances in the lactobacilli supernatants (Table 1, Fig. 3 of Supplementary Material). Chait et al. (2021) and Selis et al. (2021) found similar results when analyzing the metabolites involved in the antimicrobial activity of lactobacilli strains. Both proposed that the inhibitory activity of the supernatants was due to their acidic pH. According to our results, this effect could not be caused by the action of proteinaceous substances, since the enzymatic treatment did not suspend the inhibitory effect of any of the supernatants. However, when analyzing the ideal conditions for the action of bacteriocins produced by L.plantarum ST31, Todorov et al. (2000) found that they exerted their inhibitory action at an acidic pH. In 2022, Pei et al. found that plantaricin YKX can only form pores in the membrane of Staphylococcus aureus and promote a bactericidal effect at an acidic pH. This allows us to infer that the suspension of the antimicrobial action when neutralizing the acidic pH of the lactobacilli supernatants may have been a consequence of the alteration of the optimal pH range for the activity of molecules such as bacteriocins.

Regarding biosurfactant production, the emulsifying activity indices obtained demonstrate that L.plantarum A2 and L.plantarum 2.1 are biosurfactant-producing strains (Fig. 4). The analysis of published studies on the subject allows us to state that the emulsifying activity index of lactobacilli supernatants varies considerably depending on the lactobacillus strain and the substrate used, ranging from 7.38–84.50% (Madhu & Prapulla, 2014; Mouafo et al., 2020; Sakr et al., 2021). Furthermore, the analysis of these studies revealed that the most significant emulsifying activity indices were obtained after 24 hours of incubation (Patel et al., 2021; Sittisart et al., 2022). The emulsifying activity index demonstrated by the MRS sample (Fig. 4) was not statistically significant, and it was attributed to the presence of polysorbate 80, a biosurfactant that is part of its composition. This denotes a difference in composition and properties between MRS and the LPA2 and LP2.1 supernatants produced on the MRS medium.

Based on the results obtained so far, we have conducted a Fourier transform infrared spectroscopy (FTIR) to evaluate the functional groups present in both supernatants. The similarity between the spectra of LPA2 and LP2.1 was already expected (Fig. 5) since their performances in the assays on microplates were also similar. The verified hydroxyl groups (Table 2) can be found in biomolecules such as carbohydrates, carboxylic acids, and proteins or peptide structures (Yusof et al., 2020; Sakr et al., 2021). C-H bonds of aliphatic compounds (Table 2), in turn, are attributed to the lipid portions of the substances (Sakr et al., 2021). Furthermore, vibrations related to CH2 bonds (Table 2) are associated with lipids or regions containing proteins and lipids (Moen et al., 2005; Zoumpopoulou et al., 2010). The relationship between C = O double bonds, and waves at 1721.43 cm^− 1 (Table 2), is associated with the presence of lipid esters. While C-O single bonds, in the region of 1100 − 1000 cm^− 1 (Table 2), are associated with carbohydrates (Morais et al., 2017). The regions to which N-H bonds were provided (Table 2) are mainly related to proteinaceous compounds (Zoumpopoulou et al., 2010; Sakr et al., 2021; Sittisart et al., 2022). In view of this, the functional groups indicated by the peaks in wavelengths (Table 2) suggest that LPA2 and LP2.1 are composed of chemically diverse molecules and signal the presence of sugars, proteinaceous acids, carboxylic acids, and lipids. Furthermore, FT-IR analysis confirmed the assumption that the inhibitory effects exerted by lactobacilli supernatants are triggered by all the metabolites that compose them, in synergy (Letizia et al., 2022).

Therefore, it is possible to conclude that the LPA2 and LP2.1 supernatants consist of a complex of bioactive molecules, of different chemical natures, which may have their function impacted by changes in temperature and pH. As the identification of the biomolecules that make up the supernatants was not conducted in the present study, further research is needed, regarding molecular separation and characterization, to determine exactly which metabolites are involved in the antimicrobial and antibiofilm properties. Despite not having identified biomolecules, this study fulfilled its purpose by demonstrating that both supernatants studied are capable of antagonizing E.coli ATCC 25922 in vitro and that they have the potential to control its proliferation, maintain the balance of the microbiota during antimicrobial therapy, and reestablish the balance of the microbiota post-antimicrobial therapy.

FINAL CONSIDERATIONS

In summary, the set of data obtained by the present study attested that the supernatants LPA2 and LP2.1 can antagonize E.coli ATCC 25922 in vitro and control the proliferation of E.coli in cases of asymptomatic bacteriuria in healthy, non-pregnant women; maintain microbiota balance during antimicrobial therapy; and reestablish microbiota balance post-antimicrobial therapy. Therefore, we consider that LPA2 and LP2.1 are ready for molecular separation and characterization studies, to determine exactly which metabolites are involved in their antimicrobial and antibiofilm properties and, subsequently, for in vivo studies.

Ethics approval and consent to participate

There were no experiments with animals or that required free and informed consent from participants.

Consent for publication

All authors agree regarding the publication of the data presented.

Availability of data and material

All data generated from the research is compiled in this article and is not available on any other website.

Funding

The research was financed with resources from the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB).

Competing interests

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors' contributions

Milena Evangelista de Almeida – development of the experimental part, data analysis and writing; Maysah Meyhr D'Carmo Sodré and Samuel Santana Oliveira - support for the development of experiments; Luciana Debortoli de Carvalho, Ana Carolina Morais Apolônio and Vinícius Novaes Rocha - support for the development of microscopy experiments; Rachel Passos Rezende – support to the supervision of the work developed; Carla Cristina Romano – supervision of all experimental parts, data analysis and writing review.

Acknowledgements

Our sincere thanks to FAPESB and the Universidade Estadual de Santa Cruz for providing the necessary structure to carry out the research.

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Antimicrobial and antibiofilm activities of supernatants of Lactiplantibacillus plantarum A2 and Lactiplantibacillus plantarum 2.1 against Escherichia coli ATCC 25922

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Abstract

Background

Results

Conclusions

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1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 Conditions for growth and maintenance of microorganisms

2.2 Preparation of cell-free supernatants

2.3 Antimicrobial activity

2.3.1 Semi-solid agar diffusion

2.3.2 96-well microplate assay

2.4 Assessment of the biofilm formation capacity of Escherichia coli ATCC25922

2.5 Inhibition of biofilm formation by lactobacilli supernatants

2.5 Biofilm visualization by scanning electron microscopy

2.6 Preliminary analysis of compounds present in supernatants

2.6.2 Assessment of Biosurfactant Production

2.7 Fourier Transform Infrared Spectroscopy (FTIR)

2.8 Statistical analysis

3. RESULTS

3.1 LPA2 and LP2.1 supernatants could inhibit E.coli growth

3.2 E.coli ATCC 25922 is a moderate biofilm producer

3.3 LPA2 and LP2.1 supernatants inhibit biofilm formation by E.coli

3.4 Preliminary analysis of compounds present in supernatants

3.4.1 pH neutralization suspends the inhibitory effect of supernatants

3.5 Analysis of the functional groups that make up lactobacilli supernatants

4. DISCUSSION

Declarations

References

Additional Declarations

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