Characterization and Selection of Lactobacillus plantarum and Lactobacillus paracasei for prevention of oral bacterial infections from Chinese pickle

Jia, Guochao; Liu, Xiaofeng; Zhi, Aimin; Li, Jingjing; Wu, Yuanfeng; Zhang, Yao

doi:10.1186/s13568-021-01245-1

Original article
Open access
Published: 09 June 2021

Characterization and Selection of Lactobacillus plantarum and Lactobacillus paracasei for prevention of oral bacterial infections from Chinese pickle

Guochao Jia^1,2^na1,
Xiaofeng Liu³^na1,
Aimin Zhi²,
Jingjing Li²,
Yuanfeng Wu¹ &
…
Yao Zhang ORCID: orcid.org/0000-0001-8847-9594¹

AMB Express volume 11, Article number: 84 (2021) Cite this article

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Abstract

The oral infections were mainly caused by Streptococci and Staphylococcus aureus. Antibiotic therapies can eliminate these harmful bacteria. However, it can break beneficial microbes and lead to the persistence of resistant strains. The objective of our study was to select potential probiotic strains for the prevention of oral bacterial infections and evaluate their potential probiotic properties in oral cavity. AR113 (Lactobacillus plantarum) and AR340 (Lactobacillus paracasei) with significantly antimicrobial β-hemolytic streptococci and Staphylococcus aureus activity were isolated from Chinese pickle through agar well diffusion assay. Through the analyses of probiotic properties in antibiofilm, lysozyme and hydrogen peroxide tolerance, bacterial surface properties, adherence ability, tooth degradation and anti-inflammatory activity, the AR113 and AR340 showed anti-adhesion activity of 45.2–71.1% and 20.3–56.8% against β-hemolytic streptococci and 15.4–52.6% and 30.7–65.9% against Staphylococcus aureus, respectively, at different concentration. The two strains with high hydrophobicity, autoaggregation and survival rate adhered strongly to FaDu cells. AR113 and AR340 exhibited low calcium released from teeth (0.04 μg/mL and 0.03 μg/mL, respectively). ELISA analysis showed that AR113 and AR340 significantly inhibited the LPS-induced increase of NO and TNF-α expression. Strains-fermented skim milk inhibited the growth of β-hemolytic streptococci or Staphylococcus aureus. AR113 and AR340 were considered as probiotic candidates because of their higher antibacterial activity against some oral pathogenic bacteria, no potential of primitive cariogenicity. These candidates were expected as new probiotics with potential oral health benefits and no harmful effects.

Introduction

Oral health is an important element of general health and well-being. Although largely preventable, many people across the world still suffer unnecessarily from the pain and discomfort associated with oral diseases (Emfietzoglou et al. 2020). Oral infections constitute some of the most common forms of infections in humans (Mauramo et al. 2019). The infections of streptococcal and staphylococcal have emerged as a major source of morbidity and mortality (Peters et al. 2017). A number of distinct oral infections (e.g., angular cheilitis, parotitis and staphylococcal mucositis) are caused by these microorganism (Mccormack et al. 2015). Their impact is exacerbated by the epidemic-like emergence of resistant strains. In addition, antibiotic therapies can eliminate beneficial microbes and lead to the persistence of resistant strains (Hwang et al. 2017). New options for preventing and controlling oral infections caused by oral pathogens are urgently required.

The concept of microbial ecological change as a mechanism for preventing oral infections is important. Probiotic approach to eliminate oral pathogen is an alternative and promising way to combat infections by using harmless bacteria to displace pathogenic microorganisms. Probiotics play a pivotal role in normal body function and host health maintenance (Setbo et al. 2019; Mckenney and Pamer 2015). They alleviate the severity and duration of symptoms and reduce the incidence of oral infections (Franz et al. 2015; Kepert et al. 2016). It is shown that probiotics exhibited protective effect against various oral disorders. Probiotics can inhibit the number of Streptococcus pyogenes (Miettinen et al. 2008; Di et al. 2014), Escherichia coli (Polewski et al. 2016), Porphyromonas gingivalis (P. gingivalis) (Terai et al. 2015) and other microbial pathogens and decrease the risk of oral infections. In addition, many other beneficiary effects of administering probiotics in oral diseases have also been characterized, which includes maintenance of oral ecological balance (Jia et al. 2018), anti-inflammatory effects and immunomodulatory functions (Zupancic et al. 2017; Schmitter et al. 2018).

The probiotics play an important role in anti-oral diseases. However, evaluation criteria of probiotic properties and the mechanism in oral cavity are unresolved and needed to further research. The research described herein is part of a larger study to develop probiotics for oral health on a rational basis. The overall aim of this study is to select potential probiotic strains for the prevention of oral bacterial infections and evaluate their potential oral health benefits in vitro.

Materials and methods

Bacterial strains and culture conditions

Lactobacillus plantarum (L. plantarum) AR113 and Lactobacillus paracasei (L. paracasei) AR340 were isolated from Chinese pickle and were deposited at the China General Microbiological Culture Collection Center with preservation number CGMCC No. 13909 and CGMCC No. 15762, respectively. β-hemolytic streptococci CICC 10,373 and Streptococcus mutans (S. mutans) CICC 10,387 were obtained from the China Center of Industrial Culture Collection. Staphylococcus aureus (S. aureus) ATCC 29,213 was donated by Tongji University. Lactobacillus (LAB) strains were cultured in de Man, Rogosa and Sharpe (MRS) broth at 37 °C for 24 h. β-hemolytic streptococci strains were cultured on Columbia agar base plates supplemented with 5% sheep blood for 18 h at 37 °C under aerophilic conditions. S. aureus strains were plated onto Brian Heart Infusion (BHI) agar (Difco Laboratories, Detroit, MI, USA) for 24 h at 37 ℃.

Isolation, screening and identification of LAB

In this experiment, samples were obtained from Chinese pickle. The liquid sample (1 mL) was suspended in saline blended vigorously and centrifuged at 3000g to settle the particulate matter. Appropriate decimal dilutions were prepared and poured into sterile Petri dishes on MRS agar. The media were incubated under anaerobic conditions for 24–48 h at 37 °C (Hwanhlem et al. 2014). Colonies with different morphologies on the MRS agar plate were selected and further subcultured in order to obtain a pure colony. Glycerol stock of LAB isolates were prepared and stored at – 80 °C. The preliminary identification of strains was made by Gram staining, cell morphology and catalase reaction (Angmo et al. 2016).

The LAB strains with antagonistic activity against oral pathogen β-hemolytic streptococci and S. aureus were examined by agar well diffusion methods (Arakawa 2019). Only strains showing maximum inhibitory activity against β-hemolytic streptococci and S. aureus were selected for identification to species level. 16S rRNA of selected strains was amplified by previous PCR procedure (Angmo et al. 2016). PCR primers 27F and 1492R were employed during amplification. The sequence of PCR product was carried out by the sequencing service of Sangon Biotech Co., Ltd (Shanghai). The 16S rRNA sequences were submitted to the GenBank nucleotide sequence database under accession No. MW750439 (AR113) and No. MW750442 (AR340). Sequence results were aligned with NCBI database using BLAST algorithm. Neighbor joining method was applied to determine the closest bacterial species using MEGA software 7.0.

Antibiofilm assay

Antibiofilm properties of LAB strains against β-hemolytic streptococci and S. aureus were determined by previous method with minor modifications (Aarti et al. 2018). The culture was diluted 1:20 in medium. The 50 µL, 100 µL and 150 µL of suspensions were used to inoculate sterile 96 well polystyrene microtitre plates. After incubated for 24 h at 37 °C in 5% CO₂, wells were washed with 0.1 M phosphate-buffered saline (PBS, pH 7.2) and stained with 1% crystal violet for 15 min. The wells were rinsed again, and the crystal violet was solubilized in 200 µL of ethanol-acetone (4:1, v/v). The absorbance was read at 620 nm using ELISA reader. The percentage reduction in the biofilm formation (B) by respective pathogens was calculated as: B = [(OD_b – OD_a) / OD_b] × 100%, where OD_a is absorbance of well containing cell free neutralized supernatant and pathogens, OD_b is absorbance of well containing pathogens (Control).

Lysozyme resistance

Lysozyme resistance to assess the in vitro ability of the strains to survive in the oral cavity was performed as previous described (Garcíaruiz, 2014). LAB strains were grown in MRS broth at 37 °C for 24 h. The cells were harvested by centrifugation, washed twice with PBS and resuspended in 2 mL of Ringer solution (8.5 g/L NaCl, 0.4 g/L KCl and 0.34 g/L CaCl₂). To simulate the in vivo dilution by saliva, the bacterial suspensions (10⁷–10⁸ colonies forming units, CFU/mL)) were inoculated in a sterile electrolyte solution (0.22 g/L CaCl₂, 6.2 g/L NaCl, 2.2 g/L KCl and 1.2 g/L NaHCO₃) in the presence of 0.1 g/L of lysozyme. Bacterial suspensions without lysozyme were used as controls. Samples were incubated at 37 °C, and viable cell counts after 30 min and 120 min were enumerated on MRS agar by the drop plate method. Survival rates were calculated as a percentage of growth.

Hydrogen peroxide resistance

The ability of the selected LAB strains to grow in presence of hydrogen peroxide was studied according to method of Kullisaar et al. with modifications (Kullisaar et al. 2002). Each strain was inoculated (2%, v/v) into 10 mL MRS broth containing 3% and 6% of hydrogen peroxide, all tubes were incubated at 37 °C. After 24 h of incubation, the residual viable population was calculated by plate counting on MRS agar. Survival rates were calculated as a percentage of growth.

Bacterial surface properties

Autoaggregation capacities were performed using a previously described method (Collado et al. 2008). Hydrophobicity assays of the two selected strains were carried out using the method of Feng et al. (2017) with modifications. LAB were grown in MRS broth at 37 °C for 24 h and harvested by centrifugation (8000 g, 30 min). Cell suspension was collected and washed twice with NaCl solution (pH 7.0). Then cell suspension was adjusted OD_600nm to 0.2 by as preparation. An equal volume of dimethylbenzene was mixed into cell suspension. After mixed homogeneously for 120 s, the bacterial suspension was incubated at 37 °C for 30 min. Then, the mixture was again vortexed briefly and incubated at 37 °C for 1 h for phase separation. The aqueous phase was measured at the same wavelength. The percentage cell surface hydrophobicity (H) was calculated using the following equation: H = [(A_initial – A_final) / A_initial] × 100%, where A_initial is the initial absorbance, A_final is the final absorbance.

Adhesion to FaDu cell

The FaDu cell line was obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). FaDu cell monolayers were grown in 3-cm petri plates on microscope cover glasses until they reached confluence. Prior to adherence assays, FaDu monolayers were washed three times with PBS. Subsequently, 1 mL lactobacilli suspension (10⁷ CFU/mL in RPMI-1640) and 1 mL antibiotic-free RPMI-1640 were added to each well and incubated at 37 ℃ in a 5% CO₂ atmosphere. After 2 h of incubation, cells were washed three times with PBS, fixed with methanol, Gram-stained and then examined microscopically under oil immersion. The adherence index was evaluated in 20 random microscopic fields of adhering bacteria per 100 cells.

Adherence activity to salivary-coated hydroxyapatite

The ability of the bacteria to adhere to salivary-coated hydroxyapatite (S-HA) was measured by a previously reported method with modification (Terai et al. 2015). Briefly, human saliva was filtered through a 0.22-μm filter (Merck Millipore) after being heated at 60 °C for 30 min and centrifuged (10,000g, 10 min, 4 °C), and S-HA beads were prepared by incubating HA beads in sterilized human saliva at 37 °C for 30 min with shaking. The FITC-labelled lactobacilli or S. mutans were re-suspended in PBS to adjust the OD_550nm to 1.0. Then 5 mg S-HA beads and 2 mL bacterial cell suspension were incubated at 37 °C for 60 min with shaking. After the test tube was left for 10 min for the S-HA beads to settle, 1 mL of the collected supernatant was mixed vigorously with 0.1 mL of a 0.1 M ethylene diamine tetraacetic acid solution to dissolve the remaining HA particles. Both the OD₅₅₀ of the mixture and the control containing the bacterial cell suspension alone were measured. The adherence rate to the S-HA beads was calculated using the following formula: Adherence rate = [(OD_{550nm S. mutans} – OD_{550nm lactobacilli}) / OD_{550nm S. mutans}] × 100%. The adherence rate was used to determine whether the bacterium was adherent to S-HA.

Tooth degradation

Tooth degradation assay of the selected LAB strains was conducted (Nikawa et al. 2004). 145 mg rat teeth sample were placed in 50 ml Centrifuge tube. Each well was inoculated with 300 μL of LAB suspension (6.0 × 10⁸ CFU/mL) or S. mutans suspension (3.0 × 10⁹ CFU/mL). Subsequently, 10 mL of BHI broth was carefully added and the resulting mixtures were incubated for 0, 3, 6, 12, 24 and 48 h at 37 °C. After incubation, the amount of calcium released was measured by using a commercial kit (CA590, Leadman, Peking, China) according the manufacturer’s instructions.

Anti-inflammatory activity assay

Anti-inflammatory activity property of LAB were obtained by inhibition of nitric oxide (NO) and cytokines–tumor necrosis factor-α (TNF-α) production (Tellez et al. 2010). RAW 264.7 macrophage cells (Shanghai Institute of Life Science, Shanghai, China) were seeded on a 24-well plate at a concentration of 10⁶ cells/mL and incubated for 4 h at 37 °C in 5% CO₂. Then, the culture media was mixed with fresh media (control) or media-containing LAB cells (10⁴ cells/mL). After 2 h of incubation, lipopolysaccharide (LPS) solution (final concentration 1 μg/mL) was added and incubated for 24 h. The levels of NO and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA).

Safety assessment and enzyme activity

Hemolysin production was detected using Columbia agar plates supplemented with 5% of sheep blood (Amersco, Solon, OH, USA). The presence of α or β-hemolysis was assessed by the formation of clear or greenish zones around the colonies, respectively.

Enzyme activity was measured using the commercially-available, semi-quantitative API-ZYM system (BioMérieux, Montreal, QC) as previously described. According to the manufacturer’s instructions, cell suspension was adjusted to McFarland standards 5. Then 65 μL of cell suspension were added into each well of the API-ZYM strip and were incubated at 37 °C for 4 h in anaerobic conditions. The results were graded based on the amount of from substrate hydrolyzed on a scale from 0 (no activity) to 40 (or ≥ 40 nM).

For antibiotic susceptibility testing, LAB strains (10⁸ CFU/mL) were inoculated onto MRS soft agar. Commercial antibiotic discs (amoxicillin, penicillin, tetracycline, erythromycin, gentamicin, clindamycin, and ofloxacin, provided by Sangon Biotech (Shanghai) Co., Ltd) were placed onto the agar and incubated at 37 °C for 24 h. Resistance or sensitivity was assessed according to the CLSI/NCCLS standard.

Inhibition of Streptococci or S. aureus growth by fermented skim milk

Antimicrobial effects of the supernatant of fermented skim milk were tested against β-hemolytic streptococci or S. aureus using the agar well diffusion method (Arakawa 2019). Briefly, 100 μL of β-hemolytic streptococci or S. aureus culture (1 × 10⁶ cfu/mL) was plated onto the surface of a plate containing 20 mL of BHI agar. Then, 6-mm-diameter wells were uniformly bored in the BHI agar, and 100 μL of fermented skim milk supernatant (6000g for 15 min) was dispensed into each well. Plates were incubated at 37 °C for 24 h. After incubation, inhibition zone diameters surrounding each agar well were measured. Inhibition was considered positive when the zone diameter was > 6 mm.

Statistical analyses

All experiments were conducted in triplicate. Data analysis was performed using SPSS statistical software (SPSS, Inc., Chicago, IL). All results were expressed as mean ± SD.

Results

Isolation, screening and identification of LAB

The 32 out of 81 isolates from Chinese pickle were identified to LAB based on their Gram reaction, morphology and catalase test (data not shown). All strains were recorded as catalase negative and Gram-positive. The morphology of the strains were cocci in pairs or long chains, and bacilli in pairs or chains. Of all the isolates, two strains showed the maximal antibacterial potential against β-hemolytic streptococci and S. aureus. They were identified to be L. plantarum (AR113) and L. paracasei (AR340), respectively (Fig. 1).

Antibiofilm activity

The antibiofilm activities of selected strains against pathogens were shown in Fig. 2. Biofilm formations of pathogens were inhibited by the two strains at different concentration (50, 100 and 150 μL). The cell free neutralized supernatant of strain AR113 revealed higher anti-adhesion activity (45.2–71.1%) against β-hemolytic streptococci in a concentration dependent manner, followed by S. aureus (15.4–52.6%). However, AR340 revealed higher anti-adhesion activity (30.7–65.9%) against S. aureus in a concentration dependent manner, followed by β-hemolytic streptococci (20.3–56.8%).

Resistance to lysozyme and hydrogen peroxide

As shown in Fig. 3, survive rates of the strains in oral conditions were detected. The survival values ranged from 86.5 to 88.4% and from 76.2 to 83.4% of growth in the same medium with supplements for lysozyme and hydrogen peroxide, respectively. This result showed the high resistance of the two strains to 100 μg/mL of lysozyme under conditions stimulating the in vivo dilution by saliva and 6% of hydrogen peroxide hydrogen peroxide.