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. 2023 May 15;18(5):e0285480. doi: 10.1371/journal.pone.0285480

Characterization of the anti-pathogenic, genomic and phenotypic properties of a Lacticaseibacillus rhamnosus VHProbi M14 isolate

Jingyan Zhang 1, Kailing Li 1, Xinping Bu 1, Shumin Cheng 1, Zhi Duan 1,*
Editor: José António Baptista Machado Soares2
PMCID: PMC10184941  PMID: 37186610

Abstract

A strain of lactic acid bacteria from cheese was isolated, that showed strong growth inhibitory effects on Streptococcus mutans. The API 50CH system and 16S rDNA sequencing verified that this was a novel strain, and was named Lacticaseibacillus rhamnosus VHProbi M14. The strain inhibited the growth of S. mutans and Fusobacterium nucleatum under mixed culture conditions, coaggregated with S. mutans and F. nucleatum, and reduced the adhesion of S. mutans and F. nucleatum on cultured human primary gingival epithelial (HPGE) cells. The pH, peroxidase and protease sensitivity testing found antibacterial substances of protein- and peptide-like structures in addition to organic acids. The antimicrobial substances were sensitive to hydrolysis with trypsin, papain and pineapple protease and were inactived at temperatures above 100°C. Ammonium sulphate-precipitated proteins from the M14 strain retained the ability to inhibit the growth of S. mutans and F. nucleatum. The M14 strain contained 23 bacteriocin-related genes encoding for metabolites, belonging to class II bacteriocins. The M14 strain also showed inhibitory effects on 8 other pathogenic strains (A. actinomycetemcomitans, C. albicans, E. coli, G. vaginalis, P. acnes, P. gingivalis, S. aureus, S. enteritids), and thus has a broad spectrum of bacterial inhibition. This new isolate has been identified as having potential to be used as a probiotic bacterium in clinical applications.

Introduction

Oral diseases can be acute or chronic with a high global prevalence, and oral health is a primary component of in the maintenance of human health. Oral disease is associated with a wide variety of illnesses and disorders, including dental caries, periodontal disease, tooth loss, oral cancer [1], resulting from oral infections of some pathogenic bacteria, such as Streptococcus mutans [2, 3] and Fusobacterium nucleatum [4, 5]. A healthy oral cavity can be maintained by promoting advantageous bacteria that inhibit those bacterial species causing oral diseases [6].

Lactic acid bacteria (LAB) constitute an order of gram-positive bacteria sharing common morphological, molecular, and physiological characteristics. They are nonsporulating, non-respiring, but have aerotolerant cocci or rods, and produce lactic acid as one of the main products of carbohydrate fermentation [7]. LAB are present in the mouth, intestines, skin, and vagina of humans [8, 9], and may account for approximately 1% of the cultivable oral microbiota [10]. The most commonly isolated Lactobacilli in the oral cavity are L. casei, L. delbrueckii, L. fermentum, L. rhamnosus, and L. salivarius species [1115]. The oral cavity also contains a number of other genera, such as S. mutans, Porphyromonas gingivalis, F. nucleatum, and Prevotella intermedia [16]. These bacteria can be either pathogenic or non-pathogenic to the host, that concomitantly inhabit the oral cavity. However, with over-proliferate, microbial dysbiosis can lead to the development of a variety of oral diseases such as dental caries and periodontitis [17]. The main cause of dental caries is that S. mutans forms plaque biofilms through surface protein binding to sites on acquired membranes on the tooth surface. The inhibition and removal of dental plaque biofilm are thus important for preventing caries [18]. Probiotics can disrupt the biofilms formed by S. mutans by co-agglutinating and adhering to pathogenic bacteria [18, 19]. Recently, the antimicrobial properties of LAB and their roles in the prevention of common oral diseases have been recognized [20], with the list of the LAB species with probiotic activity rapidly increasing. Some studies have shown the beneficial effects of these probiotics in the oral cavity leading to a reduction in the risk of dental caries. For example, Zhang et al reported that L. plantarum K41 effectively inhibited S. mutans-caused biofilm formation and thus possesses a potential inhibitory effect on dental caries in vivo [21]. Nase et al. (2001) reported reduction of early childhood caries (ECC) in children consuming L. rhamnosus GG fermented milk products containing antimutans immunoglobulin G [22]. Simark-Mattsson et al. (2007) showed that L. paracasei, L. plantarum and L. rhamnosus were strong inhibitors both reference and clinical isolates of S. mutans [14]. Nikawa et al. (2004) showed that consumption of bovine milk fermented with L. reuteri was also effective in oral carriage reduction of S. mutans, resulting in a reduction in the risk of dental caries [23].

The antimicrobial mechanisms of LAB are complicated with the production of organic acids such as lactic and acetic acids from carbohydrate fermentation [20, 24]. These organic acids can enter the cells of pathogenic bacteria to reduce intracellular pH and affect cell metabolism [24]. Some bacteria such as Bifidobacterium, Lactobacillus, Lactococcus, and Pediococcus produce hydrogen peroxide and/or bacteriocins (proteinaceous compounds) that exhibit antimicrobial properties [2427]. Hydrogen peroxide is a strong oxidising agent that may rapidly penetrate the cell walls of microorganisms and inactivation the cells [28]. Bacteriocins isolated from LAB can destroy the integrity of the outer membrane of pathogenic bacteria [29].

Considering potentials of using LAB in the prevention of oral diseases, this research aimed to screen LAB strains from various sources for their capacity to inhibit oral pathogenic bacteria. We obtained 11 strains that showed significant inhibitory effects on the growth of S. mutans, and the isolate with a strong inhibition effect was identified as a novel Lactobacilli rhamnosus strain. We nominally named the bacteria L. rhamnosus VHProbi M14, and investigated its anti-pathogenic, genomic and phenotypic characteristics.

Materials and methods

Isolation and identification of LAB strains

Yoghurt, kimchi, cheese, and soya juice acquired from various retail stores and samples collected further analysis. The samples were immediately placed on ice and transported to Vland Biotech Company Laboratory, Qingdao, China. Samples (10 g) were weighed, diluted by 10 folds with normal saline, and homogenized with a paddle homogenizer (Scientz-11L, Ningbo, China) at 10 strokes/s for 5 min. The homogenate was then sequentially diluted by 10fold 3 times (10−1, 10−2 and 10−3). Diluted samples, 100 μL each, were cultured on Man-Rogosa-Sharpe (MRS) agar plates in anaerobic conditions at 37°C for 48 h. Colonies grew on the plates for 2 days, and those colonies of different shapes were selected for Gram staining and were microscopically examined. The rod-shaped and Gram-positive strains were preliminarily considered to be potential LAB candidates.

Bacteriostatic activity of the isolated potential LAB strains was then assessed using the Oxford cup diffusion agar method with some modifications as follows [21, 30]. S. mutans was initially used as the indicator strain. Firstly, 50 μL of S. mutans suspension (108 CFU/mL) was evenly spread with a spreader on brain-heart infusion (BHI) agar medium in Petri dishes. Then, Oxford cups were placed on the surfaces of the cultures, and 100 μL of the isolate strain suspension was dripped onto the surface. Finally, prepared Petri dishes were incubated at 37°C for 48 h to allow the pathogen to grow. The diameter of the inhibition zone was measured to evaluate the antibacterial activity of the isolates. All tests were performed in triplicate.

The isolates were subjected to a standard biochemical test using the API CH50 system (Biomerieux, Marcy, l’Etoile, France) and 16S rDNA sequence analysis. A large fragment of the 16S rDNA gene was amplified by PCR, using the universal primers 27F [5’-AGAGTTTGATCCTGGCTCAG] and 1492R [5’- GGTTACCTTGTTACGACTT] [31]. PCR amplification conditions consisted of preheating at 95°C for 4 min, 30 cycles of 95°C (40 s), 42°C (40 s), and 72°C (2 min), plus one additional cycle with a final 20-min chain elongation [32]. The PCR products were then purified with a Wizard PCR Preps DNA Purification System and sequenced with a Big Dye Terminator Cycle Sequencing Ready Reaction kit (BGI, China) and a model 310 automatic sequencer. The closest known relatives of the new isolates were identified by database sequence searches, and the sequences of closely related strains were retrieved from the GenBank libraries or Ribosomal Database Project databases (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree was then constructed using the NJ (Neighbor-Joining) method with MEGA 11.0 software (https://www.megasoftware.net/), and the bacterial species were identified. Those strains identified as LAB (referred to the isolate or isolates in the following context) were then used in further studies.

Pathogenic bacteria and culture conditions

Pathogenic bacterial strains tested in this study and their culture conditions are shown in Table 1. These are pathogens that can cause oral diseases, skin diseases, intestinal diseases and female gynaecological diseases, and are purchased from the BeNa Culture Collection Centre (Beijing, China).

Table 1. Pathogenic bacteria and their culture conditions.

Pathogenic bacteria Culture conditions
Aggregatibacter actinomycetemcomitans BNCC336945 Grown on Columbia blood agar (CBA) plates for 48–72 h in anaerobic conditions at 37°C
Fusobacterium nucleatum BNCC336949
Gardnerella vaginalis BNCC 354890
Porphyromonas gingivalis BNCC353909
Candida albicans ATCC10231 Grown on sabouraud dextrose agar (SDA) plates for 24 h in aerobic conditions at 37°C
Escherichia coli ATCC25922 Grown on nutrient agar (NA) plates for 24 h in aerobic conditions at 37°C
Salmonella enteritids ATCC14028
Staphylococcus aureus ATCC 6538
Propionibacterium acnes ATCC6919 Grown on reinforced clostridial agar (RCA) plates for 24 h in anaerobic conditions at 37°C
Streptococcus mutans ATCC25175 Grown on brain-heart infusion agar (BHIA) plates for 24 h in aerobic conditions at 37°C

Growth inhibition test, coaggregation test, and antagonistic adhesion test with HPGE cells

Two strains of oral pathogens (S. mutans and F. nucleatum) were used as the indicator bacteria to determine the antibacterial inhibitory properties of the isolated LAB strains.

The isolated LAB strains were scribed from glycerol tubes onto MRS agar plates and incubated anaerobically at 37°C for 48 h. Single colonies were then picked from the tubes and incubated in MRS broth medium at 37°C for 24 h. S. mutans was cultured in BHI broth medium, and F. nucleatum was cultured in BHI broth medium supplemented with 5% bovine serum.

The isolates was tested for inhibitory effect on pathogenic bacteria by co-culture in vitro according to the method described by Yang [33]. The BHI broth (10 mL) was initially mixed with 2.0% (v/v) of the isolated strain culture (109 CFU/mL) or S. mutans ATCC 25175 culture (109 CFU/mL) as the inoculants, and cultured at 37°C for 30 h. Samples were taken at 0, 4, 8, 16, 24, and 30 h, respectively, to determine the viable bacterial counts (CFU/mL) for each species. F. nucleatum was cultured in an anaerobic condition. The selective media used were respectively: MRS agar for Lactobacillus, Mitis Salivarius Bacitracin agar for S. mutans, and Differentia Clostridial Agar supplemented with 5% sheep’s blood for F. nucleatum. The distinction between colonies of F. nucleatum and the isolated strain (Lacticaseibacillus) was determined with the aid of a microscopic (Axiolab5, Carl Zeiss, Suzhou, China.). All tests were conducted in triplicate. The inhibition rate (%) was calculated as follows:

Inhibitionrate%=1CountinpathogenandM14mixture/Countinpathogencontrol×100.

The isolates were tested for their congregation capacity with S. mutans and F. nucleatum. The test was performed using a spectrophotometric assay [21, 34] with some modifications. The cells were harvested by centrifugation at 5,000×g for 15 min, washed three times with the coaggregation buffer (pH 7, containing 0.001 Mol/L Tris(hydroxymethyl)aminomethane) 0.1 mmol/L CaCl2, 0.1 mmol/L MgCl2, and 0.15 Mol/L NaCl. Cisar et al. [35], and re-suspended in the coaggregation buffer at a concentration of approximately 109 CFU/mL. The equal volumes (1 mL each) of the isolate, S. mutans or F. nucleatum suspension and a coaggregation pair (the isolate + S. mutans or the isolate + F. nucleatum) suspension were mixed, vortexed for 10 s, and incubated at 37°C for 2, 4, and 6 h precipitate the cell aggregates. The supernatant fluid (0.2 mL) was carefully removed for optical density readings (OD) at 600 nm using a spectrophotometer (Multiskan FC, Thermo scientific Shanghai, China). All tests were conducted in triplicate. The percentage of co-aggregation was calculated as follows [21]:

Coaggregationrate%=A0+B0/2Ct/A0+B0/2×100.

where A0 and B0 represent the OD values of the isolate, and S. mutans or F. nucleatum respectively at time 0, and Ct represents the OD values of the mixture 2, 4, 6 h, respectively.

Adhesion of oral bacteria to the surface of the oral cavity is required for colonization and for subsequent development of disease, therefore, prevention of adhesion of pathogens should prevent the disease [36]. In this study, human primary gingival epithelial (HPGE) cells (iCell Company, Shanghai, China) were used to determine the antagonistic adhesion of the isolated bacteria to S. mutans, or F. nucleatum. The test was performed using the Esteban-Fernández method with some modifications [37]. Adhesion tests were performed onto a 24-well chamber slide plate. The HPGE cells were seeded (2.5×105 cells/well) and incubated at 37°C in 5% CO2 for 18 h, then briefly washed twice with the medium before the following testing was undertaken.

The fresh isolated strain and pathogen (S. mutans and F. nucleatum) cultures (109CFU/mL) were washed twice with phosphate-buffered saline (PBS, pH 7.0) and suspended with the same volume of Roswell Park Memorial Institute (RPMI) 1640 culture medium that contained 10% fetal bovine serum. The OD600 absorbance was adjusted to 0.4–0.5 with the 1640 culture medium. The isolated strain and pathogen suspensions were mixed at a ratio of 1:1, and 500 μL of the mixture was added onto the prepared 24-well HPGE plate. Each treatment had three wells (repeats). HPGE cells incubated with S. mutans or F. nucleatum alone were used as the controls. The plates were incubated at 37°C in 5% CO2 for 120 min. The cells were then washed three times with PBS, fixed with methanol, Giemsa stained for 5 min, washed with PBS, and air dried. Pathogenic bacterial adhesion to each HPGE cell was observed using a microscope with a 100× oil lens (Axiolab5, Carl Zeiss, Suzhou, China.). Fifty cells in each well were observed and the pathogens adhered onto the surface of each HPGE cell was counted, and the mean for 50 counts were calculated for each well. The adhesion index was then calculated as followings:

Adhesionindex=Countofadheredpathogen/NumberofHPGEcellsi.e.,50cells.

Antibacterial test against pathogens

The antibacterial effect of the isolated strains on 10 strains of pathogenic bacteria was tested, as shown in Table 1. The isolates were grown in MRS broth at 37°C overnight. The broth was then centrifuged at 6,500×g for 5 min, and the supernatant was harvested and filtered through a 0.22μm membrane. Both the fermentation broth and supernatant were used to test their antibacterial activity against the pathogenic bacteria. Oxford cup agar diffusion assay [21, 30] as described above was used, except with the Candida albicans strain. The inhibition of the isolates of C. albicans was tested using the method described by Simark-Mattsson and Riley [14, 38] with some modifications. Firstly, 8 μL of overnight cultures of the isolated strain was spotted onto a MRS Petri dish, and incubated at 37°C for 48 h to allow some inhibitory compounds to be metabolized. Then, 7 mL of Stachybotrys glucose semi-solid medium containing 0.5% (v/v) of C. albicans (108 CFU/mL) was poured onto the plate. When the plate was solidified, the plate was then incubated anaerobically at 37°C for 24–48 h. The plate was examined regularly for presence of an inhibition circle around the colonies. All tests were conducted in triplicate.

Determination of antibacterial substances in the isolates

Many substances, such as organic acids, hydrogen peroxide and bacteriocins, produced by LAB have the ability to inhibit bacteria, so the compounds in supernatant of the cultured isolates were tested for antibacterial effects on S. mutans and F. nucleatum as followings. All tests were conducted in triplicate.

Hydrogen peroxide production

Hydrogen peroxide from the isolated strain was determined using a modified method described by Eschenbach et al. [39]. The isolate was cultured on MRS plates containing 10 g/L glucose, 0.25 g/L tetramethylbenzidine, and 0.01 g/L horseradish peroxidase (200 U/mg protein, Macklin, Shanghai, China) at 37°C in a CO2 atmosphere for 48 h. Colonies showing blue-coloured rings around the colony were classified as being H2O2 producers.

Heat sensitivity and pH

Supernatants from antimicrobial cultures grown in MRS for 48 h was divided into six aliquots. Two aliquots were adjusted with 1 Mol/L of HCl or 1 Mol/L of NaOH to pH 5.5 and 6.8 respectively. Three aliquots were adjusted with 1 Mol/L of NaOH to pH 6.8, and treated, respectively, at 80°C and 100°C for 30 min, or 120°C for 15 min [40]. The Oxford cup agar diffusion test was used to determine antibacterial activity in 100 μL of the supernatant.

Sensitivity to proteolytic enzymes

The sensitivity of the antibacterial substance in the supernatant of the isolated strains to proteolytic enzymes was tested using the method described by Hong and Wang [41, 42]. Supernatant was prepared by centrifuging the cultured isolate at 6500×g for 5 min, transferred into a clean test tube, and its pH was adjusted to 6.8 with 0.1 Mol/L sodium hydroxide. Proteolytic enzymes, trypsin (250 U/mg, Sigma, Saint Louis, American), papain (100 U/mg, Doing Higer, Ningjing, China), and pineapple protease (100 U/mg, Doing Higer, Ningjing, China) were respectively dissolved in phosphate buffer (pH 6.8) to make a concentration of 100 mg/mL. 200 uL of the enzyme preparation was then added into 20 mL of supernatant, so the final concentration of the enzyme was 1 mg/mL. The supernatant containing the enzyme was incubated at 37°C for 30 min and then at 80°C for 10 min to inactivate the enzyme. Before evaluating the antibacterial activity, the pH value was checked and readjusted to the initial pH at 6.8 with 1 Mol/L of HCl. The Oxford cup agar diffusion method was used to determine antibacterial activity in 100 μL of the supernatant.

Ammonium sulphate-precipitated proteins [43]

The supernatant was obtained by centrifuging the culture of the isolated strains at 13,000×g for 12 min. Ammonium sulphate was then added into the supernatant to achieve a saturation level of 70%, stirred slowly for 1 h and left to settle at 4°C overnight to precipitate proteins of the isolated strain. The solution was centrifuged at 13,000×g for 12 min, and the supernatant was discarded. The precipitate was dissolved in the 1/10 of the original volume of phosphate buffer (25 mmol/L, pH 6.8). The Oxford cup agar diffusion method was used to determine the antibacterial activity in 100 μL of the precipitated protein solution.

Bacteriocin production

Bacteriocin production of the isolated strains was determined using the method described by Sookkhee et al. [40] with some modifications. Briefly, the culture of the isolates strain in MRS broth (Luqiao, Beijing) under CO2 at 37°C for 48 h was centrifuged at 6,500×g for 5 min, and supernatant was collected and adjusted pH at 7.0. Supernatant was lyophilized, and the powder was then constituted in distilled water at a concentration of 600 g/L and filtered through a 0.22 μm membrane before testing against S. mutans and F. nucleatum. In the test against S. mutans, the reconstituted supernatant was firstly diluted by 2-fold using doubled BHI broth, which was then further diluted with BHI broth to the final concentrations of 150, 120, 90, 60, 30, 18, and 0 (BHI buffer only) g/L. 200 uL of each solution was added to a 96-well, flat-bottom microtitre plate, with triplicates for each concentration. Then, 2 uL of S. mutans suspension (109 CFU/mL), incubated overnight before being used, was added to each well, and 50 uL of liquid paraffin was then added carefully to the surface on to prevent moisture loss. The plates were then placed in an aerobic chamber and incubated at 37°C for 30 h. When tested against F. nucleatum, the concentrations of the reconstituted supernatant were 18, 15, 12, 9, 6, 3, and 0 g/L, and 5 uL of the F. nucleatum suspension (109 CFU/mL) was added to each well. The OD600 value (Multiskan FC, Thermo Scientific Shanghai, China) was recorded in 60 min intervals for 30 h and used to calculate the inhibition rate as follows:

Inhibitionrate%=1OD600N/OD600C×100,

where OD600N is the OD600 value for a given concentration of the reconstituted supernatant, and OD600C is the OD600 value for the control (0 g/L).

The inhibition rates and the corresponding supernatant concentrations were plotted to calculate the IC50 value using the SPSS program (IBM SPSS Statistics 20 V20.0, America).

Whole genome sequencing the isolate

The isolates, freshly cultured in broth was inoculated into 500 mL MRS broth medium at 1% (v/v) inoculum and incubated at 37°C for 22h, then centrifuged at 10,000×g for 10 min. The bacteria were collected, snap frozen in liquid nitrogen, and sent in dry ice to the Majorbio Sequencing Centre (Shanghai, China). DNA was extracted from the bacterial cultures following recommended procedures of the respective manufacturer (DP302, Tiangen, Beijing, China). A combined strategy using the Illumina Hiseq 2500 and PacBio RS II single-molecule real-time (SMRT) sequencing platforms was used to sequence the genome of this strain [44]. The genome sequences were deposited to public depository databases (https://submit.ncbi.nlm.nih.gov/). Bioinformatics analysis of the genome sequence of the strain was undertaken using the software available on the Majorbio website to determine the bacteriocin gene clusters, the composition of bacteriocin synthesis gene clusters and the type of synthesized bacteriocin. Sequence function annotations were made using NR Database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/). Secondary metabolites were analyzed using Antismash 4.0.2 software (https://dl.secondarymetabolites.org/releases/4.0.2/). Metabolic system analysis included annotation of carbohydrate-active enzymes [45] and analysis of gene clusters for secondary metabolite synthesis [46]. The sequence function was compared using BLAST+ software (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.3.0/).

Statistical analysis

All data are presented as a mean ± SD. Where applicable, a 2-tailed Student’s t-test was used to analyze the differences between treatments (Excel software; Microsoft, Redmond, WA, USA).

Results

Identification of Lactobacilli rhamnosus VHProbi M14

A total of 346 bacterial strains were isolated from different sources used in this study. Among them, 11 strains were all identified as LAB and showed significant inhibitory effects on the growth of S. mutans. The strain isolated from cheese strongly inhibited the growth of S. mutans with an inhibition zone of 2.30 ± 0.15 cm, and identified as a novel Lactobacilli rhamnosus strain. We nominally called the strain L. rhamnosus VHProbi M14 (referred to the M14 strain) and its detailed characteristics are reported here.

The M14 strain was then subject to a range of biochemical tests. The strain could ferment N-acetylglucosamine, aesculin glycerol, amidon, amygdaline, D-arabinose, L-arabinose, L-arabitol, arbulin, cellobiose, dulcitol, fructose, galactose, gentiobiose, gluconate, glucose, inositol, mannose, mannitol, maltose, melezitose, α-methyl-D-glucoside, lactose, rhamnose, ribose, salicin, sorbitol, sucrose, D-tagatose, and trehalose. The strain was defective in fermenting adonitol, D-arabitol, erythritol, D-fucose, L-fucose, glycogen, inulin, 2-ketogluconate, 5-ketogluconate, D-lyxose, α-methyl-D-mannoside, melibiose, β-methyl-D-xyloside, sorbose, turanoseraffinose, xylitol, D-xylose, and L-xylose. The results were uploaded to the API system and the strain was identified as belonging to genus L. rhamnosus. Then, the 16S rDNA sequence of the strain was uploaded to the NCBI database and the strain was found to be close to genus L. rhamnosus by BLAST comparison. The 16s rDNA sequences of 19 closely related strains were selected, downloaded, and the phylogenetic tree (Fig 1) was constructed by Neighbor-Joining method using MEGA11.0 software. The M14 strain had the highest homology with L. rhamnosus strain DM065.

Fig 1. Phylogenetic tree based on 16S rDNA sequences of Lacticaseibacillus rhamnosus VHProbi M14 and related L. rhamnosus taxa.

Fig 1

The M14 strain was thus named L. rhamnosus VHProbi M14. The 16s rDNA sequence was uploaded to the NCBI databases (https://www.ncbi.nlm.nih.gov/nuccore/OP824792).

Inhibition of the M14 strain on pathogens and antagonistic adhesion of HPGE cells

The effects of the M14 strain on the growth of S. mutans and F. nucleatum are shown in Fig 2. S. mutans in pathogen control started to grow at 4 h and reached the log phase at 8 h. The bacterial counts for S. mutans were significantly greater than those for the S. mutans + M14 treatment (P < 0.05), and the inhibition rate maintained greater than 86% from 8 to 30 h. In the inhibition test for F. nucleatum, F. nucleatum in pathogen control grew slowly from 4 to 24 h and then reached the log phase, while the bacteria on the F. nucleatum + M14 treatment did not show any growth trends. After 16 h, the inhibition rates of the M14 strain on F. nucleatum were all greater than 90%.

Fig 2. Growth curves of S. mutans and F. nucleatum and the inhibition rate (column) of the strain M14 on the growth of S. mutans and F. nucleatum.

Fig 2

(a) Growth curves of S. mutans in mixed culture (S. mutans +M14) and control culture; (b) Growth curves of F. nuleatum in mixed culture (F. nuleatum +M14) and control culture.

The M14 strain efficiently coaggregated with S.mutans and F. nucleatum, are shown in Fig 3. The coaggregation rate of the M14 strain with S. mutans and F. nucleatum increased over the period of 6 h. Coaggregation rates for strain M14 and S. mutans at 2, 4 and 6 h were 31.7% ± 3.1%, 33.8% ± 2.5% and 40.0% ± 0.1% respectively. Co aggregation rates for strain M14 and F. nucleatum at 2, 4 and 6 h were 8.1% ± 0.9%, 15.8% ± 2.7% and 21.8% ± 2.4% respectively.

Fig 3. The co-aggregation rate of L. rhamnosus VHProbi M14 with S. mutans and F. nucleatum.

Fig 3

The anti-adhesive effect of the M14 strain on adhesion of S. mutans and F. nucleatum onto HPGE cells is shown in Fig 4. As shown in Fig 4a1 and 4b1, S. mutans and F. nucleatum showed a strong attachment on HPGE cells. The adhesion index was 32.8 ± 2.54 for F. nucleatum and 10.3 ± 1.52 for S. mutans respectively. After a 2-h co-culture of the M14 strain with each of the pathogens, the adhesion index decreased to 18.93 ± 0.69 for F. nucleatum and 2.01 ± 0.67 for S. mutans, respectively (Fig 4a2 and 4b2). The reductions of the adhesion index were significant (P < 0.05). Therefore, the M14 strain showed a significant inhibitory effect on the adhesion of S. mutans and F. nucleatum onto HPGE cells.

Fig 4. The effect of L. rhamnosus VHProbi M14 on adhesion of S. mutans and F. nucleatum onto human primary gingival epithelial (HPGE) cells.

Fig 4

(a1) S. mutans control; (a2) M14 + S. mutans; (b1) F. nucleatum control; (b2) M14 + F. nucleatum.

Antibacterial test of the M14 strain

The antibacterial effects of the M14 strain on the periodontal pathogens (A. actinomycetemcomitans, F. nucleatum, P. gingivalis), the caries pathogen (S. mutans), skin pathogens (P. acnes, S. aureus), the intestinal pathogens (E. coli, S. enteritids) and vaginitis pathogens (C. albicans, G. vaginalis) are shown in Table 2. The zone of inhibition of the fementation broth ranged from 1 cm to 2.47 cm, while the zone of inhibition of the supernatant was relatively smaller. The supernatant had no inhibitory effect on C. albicans and P. acnes, presumably because these two pathogens require higher concentrations of inhibitory substances.

Table 2. Antibacterial effects (zone of inhibition, cm) of L. rhamnosus VHProbi M14 on pathogens.

Pathogenic bacteria The fermentation broth The supernatant
A. actinomycetemcomitans BNCC336945 1.00 ± 0.10 0.93± 0.06
C. albicans ATCC10231 2.10 ± 0.15 0.00 ± 0.00
E. coli ATCC25922 1.66 ± 0.18 1.17 ± 0.08
F. nucleatum BNCC336949 1.69 ± 0.09 1.60 ± 0.05
G. vaginalis BNCC 354890 1.45 ± 0.15 1.18 ± 0.03
P. acnes ATCC6919 2.47 ± 0.19 0.00 ± 0.00
P. gingivalis BNCC353909 1.40 ± 0.10 1.13 ± 0.12
S. aureus ATCC 6538 1.70 ± 0.20 1.47 ± 0.03
S. enteritids ATCC14028 1.72 ± 0.06 1.34 ± 0.11
S. mutans ATCC25175 2.30 ± 0.15 1.70 ± 0.10

Antibacterial substances in the M14 strain

The M14 strain did not show any blue-coloured ring around its colonies on plates containing 0.25 g/L tetramethylbenzidine and 0.01 g/L horseradish peroxidase, therefore, the M14 sprain does not produce hydrogen peroxide.

The inhibition capacity of the M14 strain decreased as the pH increased from 4.3 up to 6.8, but remained active at pH 6.8. This indicates the presence of other antibacterial substances in addition to organic acids (Table 3). Compared with the non-enzyme control, the proteolytic treatment of the M14 strain supernatant by trypsin, pineapple protease and papain, reduced the inhibition zone values, indicating that some of the antibacterial substances were proteins or peptides which are sensitive to the proteases. Excluding the interference of organic acids in the supernatant, the zone of inhibition around S. mutans and F. nucleatum was measured as 1.30 ± 0.05 cm and 1.39 ± 0.01 cm respectively; the inhibition zone values for the supernatant of the M14 strain culture at 80°C were 1.23 ± 0.03 cm and 1.42 ± 0.06 cm, respectively, indicating that the inhibitory substance was still active, but the antibacterial ability lost fully at temperature 100°C (Table 3). The ammonium sulphate-precipitated proteins in the M14 strain supernatant also displayed antibacterial activity against S. mutans and F. nucleatum (Table 3). These tests showed that the antimicrobial substances may consist of organic acids and protein-based antimicrobial peptides.

Table 3. Antibacterial effects (zone of inhibition, cm) of L. rhamnosus VHProbi M14 on pathogens under different conditions.

Conditions S. mutans F. nucleatum
pH pH 4.3 (Original) 1.70 ± 0.10 1.60 ± 0.05
pH 5.5 1.42 ± 0.03 1.38 ± 0.05
pH 6.8 1.25 ± 0.05 1.40 ± 0.02
Proteases (pH 4.3) Non-enzyme control 1.70 ± 0.10 1.65 ± 0.10
Trypsin 0.90 ± 0.00 1.01 ± 0.03
Papain 1.15 ± 0.05 1.21 ± 0.02
Pineapple protease 1.00 ± 0.00 1.20 ± 0.04
Temperature (pH 6.8) Control 1.30 ± 0.05 1.39 ± 0.01
80°C 1.23 ± 0.03 1.42 ± 0.06
100°C - -
120°C - -
Ammonium sulphate precipitation (pH 6.8) 1.30 ± 0.00 1.55 ± 0.05

Fig 5 shows the inhibitory effects of the reconstituted lyophilized supernatant of the M14 strain on the growth curves of S. mutans and F. nucleatum. As the growth period was extended, the reconstituted lyophilized supernatant had significant inhibitory effects on the growth of S. mutans (Fig 5a). At the 120 g/L concentration, there was no significant growth of the pathogen in 24 h. Below 120 g/L, the growth of S. mutans was inhibited to various degrees, and was depended on the supernatant concentration. S. mutans grew more slowly with increasing concentrations of the inhibitory substances. The inhibition rates were calculated separately for different concentrations at 15 hours of the growth (static period) (Table 4). The IC50 value was 58.96 g/L for S. mutans growth up to 15 h. In the case of F. nucleatum (Fig 5), there was insignificant growth of the pathogen within a 30h time period at the 15 g/L concentration. Below concentrations of 15 g/L, the growth of F. nucleatum was inhibited to various degrees, and was dependent on supernatant concentrations. F. nucleatum grew more slowly with increasing concentrations of inhibitory substances. The inhibition rates were calculated separately for different concentrations for the first 28 h of growth (static period) (Table 5). The IC50 value was 12.52 g/L for F. nucleatun for growth in the first 28 h.

Fig 5. Antibacterial effects of the concentrations of reconstituted lyophilized supernatant of L. rhamnosus VHProbi M14 on the growth of S. mutans and F. nucleatum.

Fig 5

(a) Growth curves of S. mutans; (b) Growth curves of F. nucleatum.

Table 4. The inhibition rates of the concentrations of reconstituted lyophilized supernatant of L. rhamnosus VHProbi M14 to the growth of S. mutans.

Concentrations (g/L) 0 18 30 60 90 120 150
S. mutans 0.0%±0.0% 3.1%±0.1% 12.3%±1.3% 36.0%±2.2% 82.2%±1.3% 95.7%±1.1% 98.5%±0.2%

Table 5. The inhibition rates of the concentrations of reconstituted lyophilized supernatant of L. rhamnosus VHProbi M14 to the growth of F. nucleatum.

Concentrations (g/L) 0 3 6 9 12 15 18
F. nucleatum 0.0%±0.0% 0.7%±1.1% 1.2%±1.2% 2.5%±0.8% 41.9%±0.8% 93.6%±1.2% 96.6%±1.3%

Genome annotation and bioinformatics analysis

The whole genome sequence of the M14 strain was uploaded into the NCBI database (https://www.ncbi.nlm.nih.gov/nuccore/CP095384.1/) and the total length of the genome was 2898403 bp, and could encode 2687 genes, accounting for 84.89% of the whole genome, with an average length of 915.13 bp, with a gene density of 93% and a GC content of 47.29%.

The analysis of gene clusters for secondary metabolite synthesis [43] showed that the secondary metabolites of the M14 strain included mainly SPK3-related coding genes of family I (cluster1) and cluster2 bacteriocin-related coding genes belonging to family II. There were 23 bacteriocin-related coding genes (Fig 6), with gene clusters starting at base 2320116 and ending at base 2339997 on the scaffold. The coding gene numbers ranged from 2201 to 2223, and the predicted functions of these 23 genes is shown in Table 6. Proteins ranged in size from 52 to 479 amino acids and were responsible for a range of biological functions (Table 6).

Fig 6. Bacteriocin synthesis gene cluster mapping of L. rhamnosus VHProbi M14.

Fig 6

Table 6. Details of the gene clusters for the secondary metabolite bacteriocin synthesis by L. rhamnosus VHProbi M14.

Gene ID Location Cluster ID NR Description Number of amino acids
gene2201 Chromosome cluster2 aldo/keto reductase 360
gene2202 Chromosome cluster2 threonine/serine exporter family protein 451
gene2203 Chromosome cluster2 hypothetical protein 116
gene2204 Chromosome cluster2 bacteriocin secretion accessory protein 459
gene2205 Chromosome cluster2 peptide cleavage/export ABC transporter 730
gene2206 Chromosome cluster2 GHKL domain-containing protein 431
gene2207 Chromosome cluster2 LytTR family transcriptional regulator DNA-binding domain-containing protein 258
gene2208 Chromosome cluster2 hypothetical protein 81
gene2209 Chromosome cluster2 bacteriocin immunity protein 99
gene2210 Chromosome cluster2 - 73
gene2211 Chromosome cluster2 class IIb bacteriocin, lactobin A/cerein 7B family 52
gene2212 Chromosome cluster2 MULTISPECIES: bacteriocin 61
gene2213 Chromosome cluster2 Gar-IM 105
gene2214 Chromosome cluster2 garvicin Q family class II bacteriocin 66
gene2215 Chromosome cluster2 MULTISPECIES: class IIb bacteriocin, lactobin A/cerein 7B family 52
gene2216 Chromosome cluster2 MULTISPECIES: bacteriocin 61
gene2217 Chromosome cluster2 hypothetical protein 61
gene2218 Chromosome cluster2 MULTISPECIES: hypothetical protein 268
gene2219 Chromosome cluster2 CPBP family intramembrane metalloprotease 268
gene2220 Chromosome cluster2 hypothetical protein 64
gene2221 Chromosome cluster2 Rrf2 family transcriptional regulator 146
gene2222 Chromosome cluster2 MFS transporter 475
gene2223 Chromosome cluster2 hypothetical protein 110

Discussion

The present study isolated a novel LAB strain and named it as L. rhamnosus VHProbi M14. S. mutans and F. nucleatum are the main pathogens causing dental caries and periodontal disease in the oral cavity. The M14 strain was found to inhibit the growth of the two pathogens under co-culture conditions, because the M14 strain produced organic acids or other antibacterial substances. The M14 strain efficiently co-aggregated with S.mutans and F. nucleatum. The levels of aggregation differed between the pathogens and increased with time. Not all Lactobacillus strains can aggregate with harmful bacteria [47], so the coaggregation assay may be a useful complement to screen probiotic candidates with possible anti-caries properties. These tests indicate that L. rhamnosus VHProbi M14 is a potential oral probiotic. We also found that L. rhamnosus VHProbi M14 displayed antibacterial activity on other pathogens, such as oral pathogens P. gingivalis and A. actinomycetemcomitans, intestinal pathogens Salmonella and Escherichia coli, skin pathogens P. acnes and S. aureus, and female vaginal pathogens G. vaginalis and C. albicans. Salmonella and E. coli are both pathogens that cause diarrhoea in children [48]. P. acnes is a gram-positive bacterium, found ubiquitously as a commensal on the surface of the skin, bowel, conjunctival surface, oral mucosa, and even the external ear canal. It can not only cause acne on the skin, but may also play a role in the pathogenesis of sarcoidosis and ulcerative colitis [49]. G. vaginalis is a bacterial vaginitis (BV) pathogen affecting women of childbearing age worldwide [50]. C. albicans is the most common fungal pathogen in humans, which can cause a variety of diseases such as periodontitis and fungal vaginitis [51, 52]. The ability of strain M14 to inhibit the growth of these pathogens provides theoretical support for its later application in these areas. We will further research the function in these areas.

We further investigated the characteristics of the antibacterial substances in the M14 strain using S. mutans and F. nucleatum as indicator pathogen species. The antibacterial substance in the M14 strain was free of hydrogen peroxide but consisted of organic acids and protein-based antibacterial peptides. We identified that bacteriocins (genes 2211, 2214, 2215) belonged to class II bacteriocins. Examples of class II bacteriocins include salivacin 140 from L. salivarius [20], and acidocin J1229 from L. acidophilus [53], Plantaricin EF, Plantaricin JK from L. plantarum L-ZS9 [43]. The activity of bacteriocins of class II is due to pore formation in the cytoplasmic membra [53]. The inhibitory ability of bacteriocins of class IIb is dependent on the synergistic action of the bipeptides [54]. Comparative analysis of the proteins encoded by genes 2211, 2212, 2214, 2215 and 2216 using BLAST showed that these proteins are similar to the bacteriocin proteins of strains L. rhamnosus GG, IDCC 3201, HN001 and DSM20021. LGG was able to inhibit the growth of Escherichia coli, Staphylococcus aureus, Salmonella paratyphi B and Salmonella enterica [55]. The HN001 strain can also inhibit the growth of methanogens and/or archaea bacteria [56]. Rhamnose IDCC 3201 has potent inhibitory activity against various pathogens responsible for inflammatory responses in the gastrointestinal tract (i.e. Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus and Salmonella typhi), respiratory system (i.e. Streptococcus pneumoniae) and vagina (i.e. Candida albicans) [57]. DSM 20021 strain can reduce the adhesion of pathogenic bacteria [58]. All these strains contain bacteriocin-related genes and have the ability to inhibit the growth of pathogenic bacteria. This also suggests that L. rhamnosus VHProbi M14 has the similar genetic background, as these other probiotic bacteria, to synthesize bacteriocins, consistent with M14’s antibacterial ability.

Probiotics are also gaining attention as a new type of oral care product. The function of L. rhamnosus VHProbi M14 as a potential oral probiotic will be further investigated in animal caries and periodontitis models. We will also later verify the role of this strain in the prevention and treatment of dental caries and periodontitis through clinical trials. We then expect to develop oral care products containing this strain and its metabolites.

Supporting information

S1 Table. Antibacterial effects (zone of inhibition, cm) of L. rhamnosus VHProbi M14 on pathogens.

(XLSX)

S2 Table. Antibacterial effects (zone of inhibition, cm) of L. rhamnosus VHProbi M14 on pathogens under different conditions.

(XLSX)

S3 Table. The inhibition rates of the concentrations of reconstituted lyophilized supernatant of L. rhamnosus VHProbi M14 to the growth of S. mutans.

(XLSX)

S4 Table. The inhibition rates of the concentrations of reconstituted lyophilized supernatant of L. rhamnosus VHProbi M14 to the growth of F. nucleatum.

(XLSX)

S1 Fig. Growth curves of S. mutans and F. nucleatum and the inhibition rate (column) of the strain M14 on the growth of S. mutans and F. nucleatum.

(XLSX)

S2 Fig. The coaggregation rate of L. rhamnosus VHProbi M14 with S. mutans and F. nucleatum.

(XLSX)

S3 Fig. The effect of L. rhamnosus VHProbi M14 on adhesion of S. mutans and F. nucleatum onto human primary gingival epithelial (HPGE) cells.

(XLSX)

S4 Fig. Antibacterial effects of the concentrations of reconstituted lyophilized supernatant of L. rhamnosus VHProbi M14 on the growth of S. mutans and F. nucleatum.

(XLSX)

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This study was supported by the grants from the Mountain Tai New Strategy Industry Leader Program (Grant No. tscy20180317). The person who received the financial support was Zhi Duan.

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Decision Letter 0

José António Baptista Machado Soares

8 Mar 2023

PONE-D-23-04919Characterization of the anti-pathogenic, genomic and phenotypic properties of a Lacticaseibacillus rhamnosus VHProbi M14 isolatePLOS ONE

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Please include the following items when submitting your revised manuscript:

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

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José António Baptista Machado Soares, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitled “Characterization of the anti-pathogenic, genomic and phenotypic properties of a Lacticaseibacillus rhamnosus VHProbi M14 isolate” is about to provide the characteristics of VHProbi M14 isolate.

At abstract line 10, authors specify the effects of lactic acid bacteria. For example “strong inhibitory effects” should be “strong growth inhibitory effects”.

Line19, It is not clear that the secondary metabolites is belong to bacteriocins? Bacteriocins is not a secondary metabolite. So authors should re-write that sentence.

“The M14 strain contained 23 bacteriocin-related genes encoding for the secondary metabolites, belonging to class II bacteriocins.”

Why authors mention of “bacteriostatic activity” in Line 91? It is not clear the function of bacterial suspension.

It is not clear what is the advantage of this strain compared with previously identified LAB, because the test for antibacterial activity is not referenced and compared with positive control.

Moreover, it is not sure the “Bacteriostatic activity test” I really to check the static or cidal activity.

And, What is the difference of this LAB compare with other LAB?

There are nots of unclear sentence for example “In the test against S. mutans”, “When tested against F. nucleatum,” and so on, and mistakenly written expression for example “point at line 60, OD600, CO2, IC50, membra, and sometimes missed “and”.

Strain name should be abbreviated after first time use.

References are not written consistently.

At figure 1, What is the inhibition rate by pathogenic bacteria only control mean?

Reviewer #2: In this study, the authors set out to characterize the anti-pathogenic, genomic and phenotypic properties of a novel and potential probiotic LAB. The abstract was well written, concisely overviewing the problem statement, methods, results, and conclusion. The study appears well conducted and contributes valuable data to what is already known. Before recommending it for publication, I have the following observations:

1. It is not clear how many repeated experiments (replicates) were done. Authors should include this vital piece of information.

2. For the Growth inhibition step (see line 110), authors should cite the followed method.

3. On what basis did the authors select the pathogens used in this study? Can more information about this be added to the introduction (and, if possible, the discussion) section?

4. Line 247, please change ‘346 strains of bacteria’ to ‘346 bacteria strains’

5. Given the volume of results obtained, the discussion section can be further improved, e.g. G. vaginalis is an economic pathogen affecting women of childbearing age worldwide. Authors should discuss their findings, presenting their novel strain as an alternative to treating infections caused by this pathogen.

6. The manuscript would benefit greatly if a native English speaker could proofread it for further clarity.

Reviewer #3: This manuscript describes the results of an investigation of a novel identified Lactobacillus strain with possible antimicrobial effects on various pathogens.

The topic is of interest to the scientific community as the use of probiotic strains is increasing. However a few clarifications should be made before possible publication of the manuscript.

1. Introduction: Please provide a more sufficient justification of the proposed use of the new LB strain as probiotic organism. The main problem of many infections, especially in the oral cavity, is the occurence of pathogenic biofilms. Please indicate a possible way of action of the new LB strain against established biofilms containing pathogens, as a simple application on mature biofilms will only have very little effect on them.

2. p.2, l.54: ... reported a childhood reduction of dental caries...: what is meant by this phrase? Probably you mean the reduction of eary childhood caries (ECC) in patients consuming the mentioned LB strain.

This is one of some parts of the manuscript which need a thourough revise of the language to provide a more easily readable manuscript. Further, some passages of the Materials and Methods section are quite long, please shorten this section of the manuscript and avoid repetitions.

3. Table 1: Why did you choose aerobic growth conditions for the P. acnes strain? The ATCC recommendation is an anaerobic culture for this strain (ATCC 6919, Cutibacterium acnes), please double check the correct nomenclature of all used microorganisms as these names change often.

4. Discussion: This section needs major revisions as it is mainly a repetition of the results. Please avoid any unnecessary repetitions. Some ideas for improving this section may include a discussion of possible clinical protocols for the use of the new LB strain, as well as some suggestions what further research should be carried out before an in-vivo application is possible.

Reviewer #4: The manuscript “Characterization of the anti-pathogenic, genomic and phenotypic properties of a Lacticaseibacillus rhamnosus VHProbi M14 isolate” is well written, and the study is generally presented with a good design for drawing the conclusions made by the authors. The author isolated and identified a novel LAB strain from cheese that showed the capability to inhibit a series of human pathogens including Streptococcus mutans and Fusobacterium nucleatum. The study brings new results for the LAB characterization that supports earlier studies related to the exploration of clinical probiotics. Still, the manuscript could benefit from some language polishing. I have some minor comments for changes that may be considered during a revision process. If more information is provided as described below, I recommend that the manuscript can be published in Plos One.

Introduction

Line 44: “and produce lactic acid ….”

Line 47-51: The information on other genera is irrelevant to the main context as you did not investigate these bacteria and their interbacterial relationship in the current study.

Line 60- 61: Reference.

Line 67: “in the prevention of oral diseases…”

Line 69: “a strong inhibition effect” or “the strongest inhibition effect”

M&M

The research question is well defined, relevant, and meaningful.

Line 85: where were the S. mautans strains from? Obtained from commercial providers or isolated by the authors?

Line 97: The provider of the “Big Dye TM Terminator Cycle Sequencing Ready Reaction kit”

Line 104-108: It’s good to see you have tested a wide range of pathogens that are related to various diseases. However, it would be better to provide more background information in the section of Introduction on these target pathogens.

Line 111: again, where did you get the strain of F. nucleatum?

Line 164: the reference for Oxford cup agar diffusion assay

Results

Line 247-249: what were those isolated LAB? Why did not show the result?

Line 259-260: How close? What was the similarity?

Line 271-272: “because….” This sentence should be move to the discussion section.

Line 283: reductions of the adhesion index were significant

Discussion

Line 375: these bacteriocin-related genes were…

Line 376: Did you isolate these L. rhamnosus strains?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Smith Etareri Evivie

Reviewer #3: No

Reviewer #4: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 May 15;18(5):e0285480. doi: 10.1371/journal.pone.0285480.r002

Author response to Decision Letter 0


15 Mar 2023

reposond to editor:

I have uploaded all the data as Supporting Information files.

Reposond to reviewer1:

1. Line 10, The manuscript had been revised.

2. Line 19, The manuscript had been revised.

3. Line 91, This refers to the isolates with a viable bacterial inhibitor. I have removed this expression as it may be ambiguous.

4. We screened over 300 strains from raw materials, and then screened 11 strains that could inhibit the growth of S. mutans through the Oxford Cup test . The Oxford cup inhibition test is a semi-quantitative assay. We repeated the experiment several times. We found that strain M14 had a more stable ability to inhibit the growth of S. mutans.

5. A number of inaccurate statements in the manuscript had been revised.

6. Fig 2.? In the control group, only pathogenic bacteria grew; in the experimental group, pathogenic bacteria and M14 grew together. The inhibition rate (%) was calculated as follows:

Inhibition rate (%) = (1 – Count in pathogen and M14 mixture / Count in pathogen control) ×100. Inhibition rate refers to the ability of lactic acid bacteria to inhibit the growth of pathogenic bacteria.

Respond to reviewer2:

1. All tests were conducted in triplicate. There were at least three replicates on three different occasions. The manuscript had been revised.

2. The manuscript had been revised.

3. These pathogens were chosen because of the potential application of our strains in this area and I will add some of this section to the discussion.

4. The manuscript had been revised.

5. The manuscript had been revised.

6. I will try to improve my English writing and have it revised by professionals.

Respond to reviewer3:

1. The introduction had been added and revised. The biofilm removal test and the adhesion inhibition test were also done. The biofilm test was compared by counting the number of pathogenic bacteria on the chamber slide and since the thickness of the biofilm was not measured, this test was not added to the article here.

2. Yes, the manuscript had been revised.

3. Propionibacterium acnes was cultured anaerobically. The manuscript had been revised.

4. The discussion section has been stripped of duplication and some new content has been added.

Respond to reviewer4:

1. Introduction. The manuscript had been revised.

2. M&M

Line 85: These pathogens are purchased from the BeNa Culture Collection Centre (Beijing, China) (Line 113).

Line 97: The manuscript had been revised.

Line 104-108: I will add the introduction of these pathogens in discussion.

Line 111: These pathogens are purchased from the BeNa Culture Collection Centre (Beijing, China) (Line 113).

Line 164: The manuscript had been revised.

3. Results

Line 247-249: The isolates were first observed by colony morphology and microscopy as rod-shaped bacteria. Since the main focus of this paper was on strain M14, information on other strains has been omitted.

Line 259-260: the 16S rDNA sequence of the strain was uploaded to the NCBI database and the strain was found to be close to genus L. rhamnosus by BLAST. Similar sequences refer to 16s rDNA.

Line 271-272: The manuscript had been revised.

Line 283: The manuscript had been revised.

4. Discussion

Line 375: The manuscript had been revised.

These strains are from other companies. By BLAST, they had similar bacteriocin related genes with strain M14.

Attachment

Submitted filename: Response to Reviewer4.docx

Decision Letter 1

José António Baptista Machado Soares

27 Mar 2023

PONE-D-23-04919R1Characterization of the anti-pathogenic, genomic and phenotypic properties of a Lacticaseibacillus rhamnosus VHProbi M14 isolatePLOS ONE

Dear Dr. Duan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 11 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

We look forward to receiving your revised manuscript.

Kind regards,

José António Baptista Machado Soares, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear authors,

Congratulations for the revised manuscript. Please address the minor comments added by Reviewer 1, as follow:

Please add SD in the figure and clarify the test of “Bacteriostatic activity test against other pathogens” because it was not realized a static or bactericidal activity.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I still wonder because authors said “Bacteriostatic activity test against other pathogens”, but that is just to test antibacterial effects not for whether static or cidal activity.

So, authors should change that.

Authors need to add SD in the figure.

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 May 15;18(5):e0285480. doi: 10.1371/journal.pone.0285480.r004

Author response to Decision Letter 1


29 Mar 2023

Respond to reviewer #1

In the manuscript I have replaced the expression" Bacteriostatic activity test” with "antibacterial test". The other pathogens were only tested in the Oxford Cup.

I have added SD in the figures.

Attachment

Submitted filename: Response to Reviewer4.docx

Decision Letter 2

José António Baptista Machado Soares

6 Apr 2023

PONE-D-23-04919R2Characterization of the anti-pathogenic, genomic and phenotypic properties of a Lacticaseibacillus rhamnosus VHProbi M14 isolatePLOS ONE

Dear Dr. Duan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 21 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

José António Baptista Machado Soares, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments: 

Dear authors,

Please address the minor commentors of the reviewer that I also agree, more exactly: "I still wonder because authors said “Bacteriostatic activity test against other pathogens”, but that is just to test antibacterial effects not for whether static or bactercidal activity. So, authors should change that. Authors need to add SD in the figure."

I believe that after this minor change, that the next version will be suitable for publication.

Thank you and best regards,

António

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 May 15;18(5):e0285480. doi: 10.1371/journal.pone.0285480.r006

Author response to Decision Letter 2


12 Apr 2023

This supernatant inhibition test has been done before for part pathogens. E. coli, P. acnes and S. enteritids didn’t test. I have redone the inhibition tests on these three pathogens and have added the latest test data to the manuscript. The zone of inhibition in the fermentation broth ranged from 1 cm to 2.47 cm, while the zone of inhibition in the supernatant (static or bactercidal activity)was relatively smaller. Because the bacteria in the fermentation broth are still growing and producing antibacterial substances (organic acids or bacteriocins) as it spreads.

The supernatant has no antibacterial activity against C. albicans and P. acnes, presumably because these two pathogens require higher concentrations of inhibitory substances.

I also repeated the heat sensitivity test for the inhibiting substance (excluding the interference of organic acids) and revised the manuscript.

I have added SD to the figures.

I have also revised other errors in the manuscript.

I have added a legend/caption for figures 1, 2, 3, 4, 5 and 6 in my main document.

Attachment

Submitted filename: Response to reviewer1 and editors 0413.docx

Decision Letter 3

José António Baptista Machado Soares

25 Apr 2023

Characterization of the anti-pathogenic, genomic and phenotypic properties of a Lacticaseibacillus rhamnosus VHProbi M14 isolate

PONE-D-23-04919R3

Dear Dr. Duan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

José António Baptista Machado Soares, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

José António Baptista Machado Soares

5 May 2023

PONE-D-23-04919R3

Characterization of the anti-pathogenic, genomic and phenotypic properties of a Lacticaseibacillus rhamnosus VHProbi M14 isolate

Dear Dr. Duan:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. José António Baptista Machado Soares

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Antibacterial effects (zone of inhibition, cm) of L. rhamnosus VHProbi M14 on pathogens.

    (XLSX)

    S2 Table. Antibacterial effects (zone of inhibition, cm) of L. rhamnosus VHProbi M14 on pathogens under different conditions.

    (XLSX)

    S3 Table. The inhibition rates of the concentrations of reconstituted lyophilized supernatant of L. rhamnosus VHProbi M14 to the growth of S. mutans.

    (XLSX)

    S4 Table. The inhibition rates of the concentrations of reconstituted lyophilized supernatant of L. rhamnosus VHProbi M14 to the growth of F. nucleatum.

    (XLSX)

    S1 Fig. Growth curves of S. mutans and F. nucleatum and the inhibition rate (column) of the strain M14 on the growth of S. mutans and F. nucleatum.

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    S2 Fig. The coaggregation rate of L. rhamnosus VHProbi M14 with S. mutans and F. nucleatum.

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    S3 Fig. The effect of L. rhamnosus VHProbi M14 on adhesion of S. mutans and F. nucleatum onto human primary gingival epithelial (HPGE) cells.

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    S4 Fig. Antibacterial effects of the concentrations of reconstituted lyophilized supernatant of L. rhamnosus VHProbi M14 on the growth of S. mutans and F. nucleatum.

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    Data Availability Statement

    All relevant data are within the paper and its Supporting information files.


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