Investigation of the potential probiotic effects of lactic acid bacteria and cell-free supernatants against important pathogens leading to wound infections

In recent years, several in vitro studies have revealed that some of the LAB strains and their Cell-free supernatants (CFSs) have shown antibacterial activities and promising tools to prevent or disperse the bio�lm formation of dermatological pathogens. In this study, it was aimed to investigate the antibacterial effects of LAB active cultures and CFSs against P. aeruginosa and S. aureus which were related to wound infections, as well as the inhibitory effects of CFSs on the bio�lm formation of pathogens and destroying bio�lms. The current results indicated that some of the LAB active cultures and CFSs have shown antibacterial properties against pathogens; furthermore, it was determined that some of the CFSs have shown powerful inhibitory effects on bio�lm formation and destroying bio�lms. All data together signify a great ability of the applications of CFSs as antibacterial and bio�lm removal compounds that could be very suitable to control wound infections caused by these pathogens


Introduction
Probiotics are described as living microorganisms (yeast or bacteria) that ensure the health bene ts on the host when ingested in adequate amounts [1].The most common types of microorganisms used as probiotics are lactic acid bacteria (LAB) and Bi dobacteria, as well as certain yeasts and other bacteria [2].LAB are Gram-positive bacteria include Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and have been de ned as "Generally Regarded As Safe" (GRAS) [3].
Although the gastrointestinal tract (GIT) is known to be an important resource used in the isolation of novel probiotic bacteria, it has recently been reported that attention has been focused on microorganisms found in traditional fermented foods such as fermented vegetables and dairy products.The genus Lactobacillus and Bi dobacterium are commonly found in fermented dairy products [4].
In recent studies, it has been reported that some LAB strains may be effective in the treatment of wound infections as well as many diseases.It is one of the important topics that are studied about pathogens that cause wound infections.In particular, the inhibition mechanisms are used to struggle with pathogens that cause wound infections are among the important topics studied [5].
Staphylococcus aureus is a Gram-positive bacteria that is a commensal and opportunistic pathogen.It is known to be one of the factors that cause wound infections ranging from minor infections to abscesses, from endocarditis to sepsis.Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and one of the main causes of the wound and burn infections, as well as severe diseases like cystic brosis, urinary tract infections [6].Both of these pathogens are the most common bio lm formers which remain a signi cant clinical challenge to conventional antibiotic treatment.Due to the fail of the treatment of acute and chronic wound infections and especially bio lm-related, new approaches are needed.In this regard, one of the strategies could be probiotic LAB strains which are powerful with the suppression and control of pathogens [7].Using and investigating its metabolites or structural factors as a less risky alternative to live cells has been proposed in several articles [8,9].
During the bacterial proliferation of LAB, inhibitory compounds which include organic acids, hydrogen peroxide, antimicrobial peptides, and bacteriocins may be secreted into Cell-free supernatant (CFS) and it is likely a source of antimicrobial activity against many pathogenic bacteria [10].There are very few studies investigating the antibio lm properties of CFSs [11,12].Therefore, the investigation of antibacterial and antibio lm properties of CFSs is thought to be useful in revealing the probiotic potential of LAB strains.
In this study, it was aimed to evaluate the antibacterial effects of LAB active cultures and CFSs against P. aeruginosa and S. aureus, as well as the inhibitory effects of CFSs on the bio lm formation of pathogens and destroying bio lms.

Samples and Media
The LAB strains were isolated from dairy products consisting of cheeses, yogurt, and ke r which were collected from various supermarkets and local bazaars in Aydin, Turkey.S. aureus ATCC 35556, S. aureus ATCC 25923, S. aureus 33591, S. aureus ATCC 6538, S. aureus ATCC 29213, P. aeruginosa ATCC 27853, P. aeruginosa ATCC 15692, and P. aeruginosa ATCC 9027 strains were used in this study.The strains were purchased from the American Type Culture Collection (ATCC), USA.
De Man, Rogosa, Sharpe (MRS) agar (Oxoid, England), De Man, Rogosa, Sharpe (MRS) broth (Oxoid, England), M17 agar (Merck, Germany), and M17 broth (Merck, Germany) were used to isolate and support the growth of LAB.Tryptic Soy Agar (TSA) (Merck, Germany) and Tryptic Soy Broth (TSB) (Merck, Germany) were used to support the growth of the pathogen strains.Nutrient Agar (NA) (Oxoid, England) was used for the agar well diffusion method.The pathogen strains were activated in TSB with 10% inoculum at 37 ºC overnight.All of the bacteria strains were subcultured twice before use and preserved at -20 ºC in Brain Heart Infusion (BHI) broth (Merck, Germany) containing 20% glycerol.

Isolation of LAB Strains
1 gram of the sample was aseptically transferred into the tubes containing 5 mL MRS broth and M17 broth, then the tubes were incubated at 37°C overnight in microaerobic conditions (5% CO 2 ). 1 mL of sample was added to 9 mL of sterile peptone water and 5-fold serial dilutions were prepared, 0.1 mL aliquots of the dilutions were plated onto MRS agar and M17 agar containing 50 mg/L cycloheximide.The plates were incubated at 37 °C for 48 hr in the same condition.Identi cation of the LAB strains was done based on colony morphology, Gram stain features, oxidase test, and catalase test.Representative colonies were sub-cultured on MRS agar for 48 hr in microaerobic conditions.The LAB strains were preserved at -20 ºC in MRS broth containing 20% glycerol [13].
The minimal inhibitory concentration (MIC) values of the LAB strains were determined by the Etest method.The LAB strains were adjusted to optical turbidity of the 0.5 McFarland standard (~10 8 CFU/mL) and streaked on the surface of the MRS agar.The strips of antibiotics were placed on the surface of the MRS agar and incubated in microaerobic conditions for 48 hr at 37°C [16,17].The strains were identi ed as susceptible or resistant following the cut-off values determined by EFSA (2012).

Detection of Antibacterial Properties of LAB Strains Evaluation of active cultures
Assessments of inhibitory activities of active cultures of LAB strains on the growth of the pathogens were carried out using the agar spot method, according to Leite et al. (2015) with minor modi cations [18].Brie y, the LAB strains were cultivated in MRS agar and were incubated in microaerobic conditions for 48 hr at 37°C.After incubation, their suspensions were adjusted to 0.5 McFarland standard (~10 8 CFU/mL), 2 µL of the bacterial suspensions were spotted onto the surface of MRS agar plates and the plates were incubated for 48 hr at 37°C in microaerobic conditions.The pathogen strains, which were cultivated in TSA and were incubated for 24 hr at 37°C, were adjusted to 0.5 McFarland standard (~10 8 CFU/mL).100 µL of each pathogen suspensions were added in the tubes containing 10 mL of TSB-soft agar (0.7%) and were poured into petri dishes of LAB strains.The petri dishes were incubated for 24 hr at 37°C in aerobic conditions.The inhibitory effects of active cultures were evaluated by measuring the diameter of the inhibition zone around each spot of the LAB strains [18].
Evaluation of cell-free supernatants (CFSs) Assessments of antibacterial activities of CFSs were carried out using the agar well diffusion method, according to Shokryazdan et al. (2014) with minor modi cations [19].Brie y, overnight cultures of the LAB strains were inoculated into (1%, v/v) MRS broth and were incubated at 37°C for 24, 48, and 72 hr.The pH of the strains after incubation was measured with a pH meter.Then, CFSs were prepared by centrifuging the broth at 4000 x g for 20 min at 4 °C.The CFSs were ltered with a 0.22-μm syringe lter (Millipore, Bedford, MA, USA).
Each pathogen strain, which was cultivated in TSA and incubated at 37°C overnight, was adjusted to 0.5 McFarland standard (~10 8 CFU/mL).The pathogens were streaked on NA plates with a cotton swab and the petri dishes were allowed to dry at room temperature.A well (6 mm in diameter) was cut using a sterile cork borer, and then 100 μL of the CFS was pipetted into each well.The petri dishes were incubated at 37°C overnight.To investigate whether the CFS inhibited the growth of the pathogen, the CFS of the selected LAB strain was adjusted to pH:7 using NaOH and was neutralized and that is called Neutralized Cell-free supernatant (NCFS).The antibacterial activities of CFS and NCFS were evaluated separately [19].

Detection of Antibio lm Properties of LAB Strains
The bio lm formations of pathogen strains were tested according to incubation time at 37°C for 48 hr using the microtiter plate method as described by Ersoy et al. (2019) with minor modi cations [20].Total bio lm biomass was measured and evaluated [21].

Pre-incubation experiments
The inhibitory effects of CFSs on the bio lm formation by pathogens were tested with the pre-incubation experiments which were carried out as described by Gudiña et al. (2010) and Bulgasem et al. (2015) with minor modi cations [22,23].Brie y, 200 μL of CFS were pipetted into each well and the plate was incubated at 37 °C overnight.The pathogen strains were cultured in TSB and 1/40 dilution of the overnight culture containing 0.25 % glucose (Sigma-Aldrich, USA) was prepared.200 μL of the pathogen suspension were inoculated into each well after the CFS was removed.200 μL of un-inoculated TSB containing 0.25 % glucose and 200 μL of un-inoculated MRS broth were used as negative controls.200 μL of diluted pathogen suspension was used as a positive control for bio lm growth.The plates were incubated at 37°C for 48 hr.Following the day, the media was aspirated and the non-adherent cells were removed by washing the wells three times with 200 μL of PBS (Sigma-Aldrich, USA).1% crystal violet solution (CV) (Merck, Germany) was pipetted into wells (200 μL/well) and incubated for 15 min.After incubation, the wells were washed three times and the plates were allowed to dry at room temperature.80% ethanol-20% acetone (Sigma-Aldrich, USA) were pipetted into wells (200 μL/well) and the optical density (OD) of each well was measured at 595 nm using a microplate reader (Thermo Fisher Scienti c, USA).Each sample was analyzed in triplicate.The reduction of total bio lm mass was evaluated as described by [24].

Co-incubation experiments
The inhibitory effects of CFSs on bio lm formation were also tested with the co-incubation experiments were carried out as described by Gudiña et al. (2010) and Bulgasem et al. (2015) with minor modi cations [22,23].Brie y, the pathogen strain (100 µL) and CFS (100 µL) were pipetted into each well and the plate was incubated at 37 °C for 48 hr.The microtiter plate method and CV staining method, which were described in pre-incubation experiments, were also used in this section.In addition to negative controls, 100 μL of un-inoculated MRS broth + 100 μL of TSB containing 0.25 % glucose were used as a negative control for mixed solutions of the pathogen and CFS.
Each sample was analyzed in triplicate.The reduction of total bio lm mass was evaluated [24].

Post-incubation experiments
The post-incubation experiment was carried out as described by Zamani et al. (2017) with minor modi cations [25].Brie y, the pathogen strains were allowed to grow in wells (200 μL/well) at 37 °C for 48 hr to form bio lms, and then CFSs were added to these wells (200 μL/well), and the plate was incubated at 37 °C overnight.The microtiter plate method, CV staining method, and OD measurements were used as described before.

Statistical analysis
Quantitative data were described by using mean values and standard deviation (SD) from three independent experiments.A negative and positive control samples were used for each assay.Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS) version 22.The results of all bio lm experiments were statistically analyzed using two-way ANOVA followed by Tukey's post hoc test.A pvalue ≤ 0.05 was considered signi cant.

Antibiotic Susceptibility Pro le of LAB Strains
The LAB strains were screened for antibiotics resistance.Clindamycin, gentamycin, streptomycin, tetracycline, and vancomycin were recommended by EFSA (2012).The strains were categorized as susceptible or resistant following the cut-off values.C. alimentarius HBUAS52461, L. delbrueckii subsp.bulgaricus ND04 ve L. lactis IL6288 were only evaluated for vancomycin and were determined susceptible to other antibiotics.According to the EFSA document, other strains, which have intrinsic resistance to vancomycin, were not required to be evaluated for vancomycin and they were also found susceptible to other antibiotics.

Evaluation of cell-free supernatants (CFSs)
When the antibacterial activities of CFSs were evaluated, L. delbrueckii subsp.bulgaricus ND04 and L. mesenteroides subsp.mesenteroides CAU4374 were found to be effective against only P. aeruginosa strains.P. parvulus 2.6, L. paracasei RV-M192, L. paracasei ZFM 54 and L. coryniformis MXJ 32 were also found to be effective against all pathogens.Almost all of the highest inhibition zone diameters were determined after the CFS-72 application among CFSs and there were no inhibition zones following the NCFS (Table 3).
Detection of Antibio lm Properties of LAB Strains P. aeruginosa and S. aureus strains were able to form a bio lm throughout the 48 hr of incubation.S. aureus ATCC 33591 strain was moderate and all other pathogens formed strong bio lm producers.S. aureus ATCC 35556, S. aureus ATCC 6538, P. aeruginosa ATCC 27853, and P. aeruginosa ATCC 15692 strains were selected for pre-incubation, co-incubation, and post-incubation experiments.

Co-incubation experiments
Another experiment was conducted to evaluate the possible properties of CFSs to inhibit the formation of bio lms.It was determined that CFS-72 of L. lactis IL6288 for P. aeruginosa ATCC 15692 (96%) and P. aeruginosa ATCC 27853 (95.7%),CFS-72 of L. reuteri SKB1241 for S. aureus ATCC 35556 (94.2%) and CFS-72 of L. paracasei RV-M192 for S. aureus ATCC 6538 (96.2%) have shown the highest bio lm inhibition rates (Fig. 3A).It was determined that the bio lm formation potentials of the pathogens were signi cantly inhibited by NCFS-72 (Fig. 3B).These results were statistically signi cant (p <0.05).

Discussion
Recently, many studies are searching for new alternative antimicrobial therapy such as natural product-based therapies.In this context, most researchers have focused on the use of probiotics as candidates for natural product-based therapies [7].There are couple of important studies which highlight that LAB species are well-established probiotics and have antibacterial and antibio lm activities [11,26] which can be evaluated in dermal applications [27] and murine wound infection models [28].Overall, LAB has been reported as a promising tool to struggle with many pathogens.Therefore, it is important to display the LAB strains and determine their bene cial and functional features as a probiotic candidate.According to this idea, it was aimed to investigate some of the probiotic properties of novel LAB strains and their CFSs against some strains of S. aureus and P. aeruginosa that were related to wound infections.
Dairy products including cheese, lor cheese, çökelek, tulum cheese, yogurt, ke r are known as good sources of probiotics which include the genera Lactococcus, Lactobacillus, Leuconostoc, and Pediococcus [29].Pediococcus is one of the starter cultures that can be used in the fermentation of dairy products.Many Pediococcus strains produce bacteriocins (i.e., pediocins) that have antibacterial properties against many pathogens [30].In this study, P. parvulus 2.6 was isolated from white cheese.The genome sequence has been determined by Pérez-Ramos et al. ( 2016) and probiotic properties were evaluated by [31].Immerstrand et al. (2010) reported that P. parvulus 2.6 is a preservative strain that inhibits the growth of the sporulating food pathogen and spoilage bacterium Bacillus cereus.L. delbrueckii subsp.bulgaricus ND04 was isolated from çökelek cheese in the present study [32].Active culture and CFS of this strain were found to have an inhibition effect only on P. aeruginosa according to the agar spot and well diffusion methods.L. paracasei ZFM 54 was isolated from yogurt in the present study.The genome sequence analysis and in vitro probiotic characteristics were reported before.Probiotic characterization and anti-Helicobacter pylori activities were determined by Qureshi (2020) [33].It was reported that this strain has numerous probiotic-associated genes and characteristics.L. lactis is a starter culture that uses dairy products like yogurt and cheese.It can be used in therapeutic utilization for human and animal health due to being a probiotic bacterium.In this study, L. lactis IL6288 was isolated from tulum cheese.It was completely sequenced before [34].L. coryniformis MXJ 32 was isolated from white cheese and found to have inhibitory effects on all pathogens in the agar well and agar diffusion methods.Lü et al. (2014) reported that L. coryniformis MXJ 32, which has both bacteriocin-producing features and a broad antimicrobial spectrum, was isolated from a traditional fermented vegetable (Jiangshui Cai) [35].
Many studies have reported that there are various in vitro methods to evaluate the antimicrobial activities of CFSs.The agar spot and agar well diffusion methods were described to use by several authors [36].Similar to other studies, they were preferred in this study.It was found that the antibacterial activities of active cultures and CFS-72 of L. delbrueckii subsp.bulgaricus ND04, L. mesenteroides subsp.mesenteroides CAU4374 against P. aeruginosa and P. parvulus 2.6, L. paracasei RV-M192, L. paracasei ZFM 54, L. coryniformis MXJ 32 against all pathogenic bacteria were similar.
This study has also shown that the pH values of CFSs were between 4 and 5.5 before the neutralization process.To eliminate the effects of organic acid molecules including; lactic, acetic, and formic acid or bacteriocins selected CFSs that inhibit the growth of pathogens were neutralized.After the neutralization process, the loss of inhibition effects of tested NCFSs on pathogens have shown that the inhibition effects could arise from the production of organic acid molecules or bacteriocins that were active under acidic conditions only.Koohestani et al. (2018) reported that after the neutralization process, the antibacterial effects of CFSs of L. casei 431 and L. acidophilus LA5 strains on the pathogen S. aureus ATCC 25923 were lost and the effect was caused by organic acid production [37].Boris et al. (2001) found that some of the lactobacilli strain dramatically hindered the growth of S. aureus and P. aeruginos because of organic acid production [38].Layus et al. (2020) investigated the antibacterial effect of L. plantarum CRL 759 on P. aeruginosa and methicillinresistant S. aureus (MRSA) [9].Research has shown that CFS (SLp759) inhibited the growth and adhesion of pathogens.According to high-performance liquid chromatography (HPLC) analysis, the inhibitory effect was found due to the production of organic acids.
Many inhibition mechanisms, which are secretion of antagonistic substances (e.g., bacteriocins, exopolysaccharides (EPS), organic acids, hydrogen peroxide), and unfavorable conditions for pathogens (e.g., competition for surface and nutrients), have been reported in several studies [39].It was also reported in several studies that long-term cultivation of LAB strains may have a strong inhibition effect on pathogenic bacteria due to produce carbon dioxide (CO 2 ) while growing.Some researchers indicated that supernatants extracted from ve-day [12] and 3-day cultures [40] were utilized to allow for the maximal accumulation of antimicrobial compounds.In this study, when the inhibitory activities of CFSs, which were obtained from 24, 48, and 72 hr incubation, were compared, the highest inhibitory activities were found CFS-72.The antibacterial activity could be due to long-term cultivation.
In recent years, several studies have revealed that screening of the antibio lm activities of CFSs has been evaluated by different methods .The results of the pre-incubation experiments proved that CFS-24 of L. paracasei ZFM 54, CFS-72 of L. delbrueckii subsp.bulgaricus ND04, CFS-72, and CFS-48 of L. coryniformis MXJ 32 have shown the highest bio lm inhibition rates respectively, P. aeruginosa ATCC 15692, P. aeruginosa ATCC 27853, S. aureus ATCC 35556, and S. aureus ATCC 6538.Unlike the test pathogens used in this study, researchers reported that CFSs of the LAB strains inhibited the adhesion of ve Candida spp.bio lm in pre-incubation and coincubation experiments [23].
It has been reported that CFSs of some lactic acid bacteria decrease the adhesion of pathogens to the surface and made the surface more hydrophilic.Bacteriocins, which are one of the examples of them, can change the physical and chemical conditions that affect bio lm formation [25,41].Koohestani et al. (2018) evaluated the bio lm destructive properties of CFSs of L. casei 431 and L. acidophilus LA5 on the pathogen S. aureus ATCC 25923, and they reported that CFSs could be a potential therapeutic agent in removing bio lm structures from both hydrophobic and hydrophilic surfaces [37].
In the present study, CFS-72 of L. reuteri SKB1241 for S. aureus ATCC 35556, L. lactis IL6288 for P. aeruginosa ATCC 27853 and P. aeruginosa ATCC 15692 and CFS-72 of L. paracasei RV-M192 for S. aureus ATCC 6538 were determined the highest bio lm inhibition rates in the co-incubation experiment.Khiralla et al. (2015) reported that CFSs of L. pentosus and L. plantarum signi cantly reduced bio lm formation of P. aeruginosa in the co-incubation experiment [42].The effect of antibio lm was explained as the inhibition of the adhesion to the microplate surface or the inhibition of quorum sensing (QS) of pathogens by organic acids, bacteriocin, and other inhibitory substances in the supernatant rather than inhibiting the growth of pathogens.In this study, CFS-72 of L. lactis IL6288 was found to inhibit bio lm formation of P. aeruginosa ATCC 27853 and P. aeruginosa ATCC 15692.After neutralization of the acidic pH of the CFS, it was determined that inhibitory activity was still observed against these pathogens.Sikorska and Smoragiewicz (2013) reported that some of the LAB strains including L. reuteri, L. rhamnosus GG, L. paracasei and L. casei inhibited MRSA bio lm formation [43].The authors demonstrated that the mechanism may be related to the contest and presence of acids and/or bacteriocin.
The results of this study proved that CFS-72 of L. reuteri J33, L. delbrueckii subsp.bulgaricus ND04 and L. lactis IL6288 for P. aeruginosa ATCC 15692, CFS-72 of L. mesenteroides subsp.mesenteroides CAU4374 for S. aureus ATCC 35556 and P. aeruginosa ATCC 27853, CFS-48 of L. delbrueckii subsp.bulgaricus ND04 for S. aureus ATCC 6538 have shown the highest inhibition rates for destroying bio lm in the post-incubation experiment.Hor and Liong (2014) reported that the CFS of L. bulgaricus FTDC 8611 dramatically inhibited the bio lm formation of S. aureus by which the organic acids [44].The current results indicated that CFS-48 and CFS-72 of L. delbrueckii subsp.bulgaricus ND04 have shown high antibio lm potential against P. aeruginosa ATCC 15692 and S. aureus ATCC 6538, respectively.El-Mokhtar et al. ( 2020) evaluated the antibio lm potential of CFSs of L. fermentum, L. paracasei, L. bulgaricus, L. plantarum, L. acidophilus strains against P. aeruginosa pathogens [24].CFSs were used both co-and post-incubation experiments.It has been reported that the capability of CFS to hinder bio lm structures may be caused by the presence of bio-surfactants and exopolysaccharides.Zamani et al. (2017) isolated L. plantarum strains from various cheeses and investigated the antibio lm effects of CFS on P. aeruginosa and S. aureus pathogens [25].Researchers reported that CFS both decreased the bio lm formation by pathogens and disrupted preformed bio lm structures.It was also determined that the highest activity of CFSs was on P. aeruginosa pathogen.Rana et al. ( 2020) investigated the antibio lm potential of CFSs (acidic and neutralized) of some of the lactic acid bacteria strains in P. aeruginosa PAO1 (MTCC 3541) using co-treatment and post-treatment methods [11].Researchers reported that CFSs have shown signi cant inhibition of bio lm formation.
In this study, after the neutralization process according to the bio lm experiments, the inhibition effect was not observed in some, while it was found to be reduced, unchanged, or remained almost the same in others.The inhibition effects may be multifactorial, and included much more than the antibacterial organic acid molecules and\ or bacteriocin-like molecules.
Considering that S. aureus and P. aeruginosa are important species encountered in the treatment of acute and chronic wounds, the application of CFSs as antibacterial and bio lm removal compounds could be very suitable to control wound infections caused by these pathogens.

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Table 1
Molecular identi cation of the LAB strains

Table The
antibacterial effects of CFSs