Antimicrobial activity of Lactiplantibacillus plantarum APsulloc 331261 and APsulloc 331266 against pathogenic skin microbiota

1. Abstract 2. Introduction 3. Materials and methods 3.1 Bacteria and fungi strains, and culture conditions 3.2 Indirect co-culture assay 3.3 Cell culture with CM assay 3.4 Plate agar overlay assay 3.5 DNA isolation, PCR, and agarose gel electrophoresis 3.6 Statistical analysis 4. Results 4.1 APsulloc 331261 and APsulloc 331266 inhibit the growth of S. aureus 4.2 APsulloc 331261 and APsulloc 331266 suppress the growth of C. acnes 4.3 The growth of C. albicans was inhibited by APsulloc 331261 and APsulloc 331266 4.4 The growth of M. globosa andM. restricta was suppressed by APsulloc 331261 and APsulloc 331266 5. Discussion 6. Conclusions 7. Author contributions 8. Ethics approval and consent to participate 9. Acknowledgment 10. Funding 11. Conflict of interest 12. References


Abstract
Balanced skin microbiota is crucial for maintaining healthy normal skin function; however, disruption of the balance in skin microbiota is linked with skin diseases such as atopic dermatitis, acne vulgaris, dandruff, and candidiasis. Lactoplantibacillus species with proved with health benefits are probiotics that improve the balance of microbiome in skin and gut. In the present study, we investigated the potential antimicrobial activity of Lactiplantibacillus plantarum APsulloc 331261 (APsulloc 331261) and Lactiplantibacillus plantarum APsulloc 331266 (AP-sulloc 331266) derived from green tea, in inhibiting five skin pathogenic strains (Staphylococcus aureus (S. aureus), Cutibacterium acnes (C. acnes), Candia albicans (C. albicans), Malassezia globosa (M. globose), and Malassezia restricta (M. restricta)) associated with skin infection. Viability of S. aureus, C. acnes, C. albicans, M. globosa, and M. restricta was inhibited by indirect co-culture with APsulloc 331261 or APsulloc 331266 at various ratios. Different concentrations of the cell-free conditioned media (CM) derived from APsulloc 331261 or APsulloc 331266 inhibited the vaibility of S. aureus, C. acnes, C. albicans, M. globosa and M. restricta in a dose dependent manner. Moreover, susceptibility of S. aureus, C. acnes, and C. albicans against APsulloc 331261 or APsulloc 331266 was confirmed following agar overlay methods. Results of the agar overlay confirmed that various concentrations of APsulloc 331261 and APsulloc 331266 exhibited low to high inhibitory activity on the growth of S. aureus (ZDI 20.3 ± 2.1-32.3 ± 2.1 mm, R value 5.7 ± 0.8-7.8 ± 1.3 mm), C. acnes (ZDI 15.0 ± 1.7-22.2 ± 1.7 mm, R value 3.2 ± 1.3-5.5 ± 1.3 mm) and C. albicans (ZDI 13.3 ± 4.0-27.0 ± 3.6 mm, R value 2.8 ± 1.9-5.5 ± 1.7 mm). Finally, standard PCR analysis identified the presence of the of plantaricin genes encoding antimicrobial peptides in APsulloc 331261 and APsulloc 331266. These results suggest that APsulloc 331261 and APsulloc 331266 has a potential effect in the improvement of the balance of skin microbiota by inhibiting skin pathogenic strains.

Introduction
Probiotics are defined as living organisms that provide a health benefit on the host when administered in adequate amounts [1]. Probiotics are gaining immense attention in scientific research, suggesting potential health benefits to host organism [2,3]. Lactic acid bacteria (LAB) are the main bacteria that produce lactic acid as a major fermentation product using carbohydrate, and Lactoplantibacillus (formerly Lactobacillus) [4] species abundantly being presented in the intestines of vertebrates form the major part of this group [5]. Lactiplantibacillus plantarum (L. plantarum) with proved health benefit based on well-designed clinical studies are used probiotics in the food and nutraceutical industries [6,7]. L. plantarum are known to produce various antimicrobial compounds such as bacteriocin, hydrogen peroxide, and acids [8]. Bacteriocins are ribosomally synthesized antimicrobial peptides that inhibit or kill the bacterial strains closely related or non-related to produced bacteria without causing any harm to bacteria themselves owing to the presence of self-defensive proteins [9,10].
The skin possesses diverse microbiome, which provides protection against invading pathogens by the production of protein complex antibiotics [11,12]. Change in the homeostasis between the host and microorganism is associated with skin diseases or infections [12]. A variety of key microbial genera are associated with skin health, including bacterial species Staphylococcus and Cutibacterium (formerly Propionibacterium), and fungal species Candida and Malassezia. Staphylococcus aureus (S. aureus) is a gram-positive and facultative anaerobic bacteria [13]. The colonizationof S. aureus in the skin have an important role in the pathogenesis of atopic dermatitis (AD) by disrupting the epidermal barrier and contributing to flare and itching by inducing degranulation of mast cells [14]. Cutibacterium acnes (C. acnes), a gram-positive and aerotolerant anaerobe bacterium, is the primary trigger for opportunistic infection, aggravating acne lesions [15]. The breakout of acne occurs in the sebaceous gland in the skin where increased sebum secretion promotes abnormal keratinisation, resulting in follicular clogging and proliferation of C. acnes. Subsequently, it induces inflammatory cytokine responses, leading to redness and oedema, and exacerbates acne symptoms [16,17]. Candidiasis is fungal infection caused by Candida and occurs in the skin, fingernails, mucous membranes throughout the body. Candida is a part of fungal commensals in the human body; however, when the immune system of the body becomes compromised or the skin is broken, Candida can cause infections in the skin, fingernails, mucous membranes throughout the body [18][19][20]. Malassezia is a lipophilic fungal genus resident on normal skin and observed in approximately 75%-98% of healthy adults [21]. Malassezia generated free fatty acids, particularly lipase mediated breakdown of sebum triglycerides, can induce flaking in dandruff-susceptible patients [22].
Antibiotics administration to treat skin diseases may result in negative effects, such as dose-dependent toxicity, and the emergence of resistant pathogenic strains [21,23,24]. Infections caused by antibiotic resistant bacteria are serious threat to global health. The pathogens causing these infections can obtain the antibiotic resistance genes from proliferation of multi-drug-resistant bacteria, which, in turn, abrogate the effect of antibiotics due to persistence of the resistance genes in the microbiota [25]. Topical or oral administration of probiotics, such as Lactiplantibacillus species, could ameliorate skin health by inhibiting colonisation of skin pathogens and maintaining the balance of the gut flora [26][27][28]. Therefore, there is an urgent need to develop innovative strains of probiotics for alternative antimicrobial therapy against infectious diseases.
The safety and beneficial effect of Lactiplantibacillus plantarum APsulloc 331261 (APsulloc 331261) and Lactiplantibacillus plantarum APsulloc 331266 (APsulloc 331266) isolated from green tea (Camellia sinensis) have also been described [29]. APsulloc 331261 alleviates gastric inflammation, which might be attributed to the production of propionate derived from the modulation of gut microbiome in an alcohol-dependent gastric ulcer [30]. APsulloc 331261 significantly boosts the abundance of various intestinal beneficial bacteria such as Bifidobacterium spp. and Clostridium butyricum. Moreover, APsulloc 331261-derived extracellular vesicles exerts beneficial effects on human skin by inducing anti-inflammatory cytokine-mediated by M2 macrophage polarisation [31]. In the present study,we examined the antimicrobial activity of APsulloc 331261 and APsulloc 331266 against five skin pathogenic strains (S. aureus, C. acnes, C. albicans, M. globosa and M. restricta). To determine antimicrobial activity of APsulloc 331261 and APsulloc 331266, we validated the viability of five strains implicated in skin disease by indirect co-culture with APsulloc 331261 or APsulloc 331266, and cell-free conditioned media (CM)-derived from APsul-

Bacteria and fungi strains, and culture conditions
APsulloc 331261 (KCCM11179P) and APsulloc 331266 (KCCM11180P) isolated from green tea derived from the leaves of Camellia sinensis (Osulloc farm, Jeju island, South Korea) [29] were cultured in MRS broth at 37 • C for 24 h (Becton, Dickinson and Company, Sparks, MD, USA). The bacterial and fungal strains were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) (listed in Table 1) and cultured according to ATCC instructions. S. aureus and C. albicans were cultured in tryptic soy (TS) broth (Becton, Dickinson and Company) and yeast malt (YM) broth (Becton, Dickinson and Company), respectively, at 37 • C, on a shaking incubator, for 24 h. M. globosa and M. restricta were cultured in modified Leeming & Notman agar (MLNA; ATCC medium 2737) without agar on a shaking incubator at 30 • C for 72 h. C. acnes was cultured in modified reinforced Clostridium (MRC) medium (ATCC medium 2107) at 37 • C for 72 h under anaerobic conditions. The cultures were diluted 1:50 or 1:100 in the indicated media, sub-cultured to mid-to-late logarithmic phase and adjusted to an OD 600 of 0.5 before use.

Indirect co-culture assay
Five pathogenic strains (S. aureus, C. acnes, C. albicans, M. globosa and M. restricta) were co-cultured in APsulloc 331261 or APsulloc 331266 using a 6-well transwell chamber with a 0.4 µm porous membrane (Co-star, Corning, NY, USA) [32,33]. The total volume of a well was consisted of 5 mL broth. 0.2% (v/v) of the pathogens (OD 600 of 0.5) was placed in the basolateral compartment of transwell. S. aureus and C. albicans were incubated with MRS broth at 37 • C for 48. C. acnes was incubated in MRC broth at 37 • C for 96 h using BD GasPak™ EZ container systems (Becton, Dickinson and Company). M. glo-bosa and M. restricta were incubated with MLNA medium without agar at 30 • C for 72 h. Various ratios (v/v) of the pathogens to APsulloc 331261 and APsulloc 331266 (1:0.001, 1:0.01, 1:0.1 and 1:1) were inoculated in the insert with the indicated media used for incubation of pathogens to determine antimicrobial activity. The pathogenic culture without inoculation of APsulloc 331261 and APsulloc 331266 was used as control. After incubation, the viability of the pathogens was measured at OD 600 using a microplate reader (SpectraMax i3x, Molecular Devices, San Jose, CA, USA).

Cell culture with CM assay
To obtain the cell-free CM, overnight culture of APsulloc 331261 and APsulloc 331266 was sub-cultured to mid-to-late logarithmic phase and adjusted to OD 600 of 1.0. APsulloc 331261 or APsulloc 331266-derived supernatant was obtained after two centrifugation steps at 15000 × g for 10 min. Supernatants were sterilized using 0.45 µm and 0.2 µm syringe filters (Whatman™, Maidstone, UK) and stored at -20 • C. Prepared CM (100%) was diluted to various concentrations (75%, 50%, 25%, 10% and 5%, (v/v)) with MRS broth and incubated with 0.2% (v/v) of five pathogenic strains in 6 well plates at 37 • C for 48 h, except on C. acnes for 96 h. The viability of the pathogens was measured at OD 600 using a microplate reader.

Plate agar overlay assay
The effects of APsulloc 331261 and APsulloc 331266 on the growth of S. aureus, C. acnes, and C. albicans were examined on agar plates as previously described [32]. For the plate agar overlay assays, 1, 2 and 4 µL of APsulloc 331261 and APsulloc 331266 adjusted to an OD 600 of 0.5 (ca. 1 × 10 4 , 2 × 10 4 , and 4 × 10 4 colony forming unit (CFU)) were spotted on 1.5% MRS agar plates and incubated for 48 h at 37 • C. After cooling to 40 • C in an autoclaved agar medium, S. aureus, C. acnes, and C. albicans suspension in 0.7% soft agar (TS, MRC, YM, respectively) were prepared and overlaid on the plates incubated with APsulloc 331261 and APsulloc 331266. After the plates were incubated at 37 • C for 24 h under aerobic (S. aureus, C. albicans) or anaerobic (C. acnes) incubators, the zone of diameter inhibition (ZDI) value was determined following the method adopted by Shokryazdan et al. [

DNA isolation, PCR, and agarose gel electrophoresis
Total DNA from APsulloc 331261 and APsulloc 331266 was extracted using a G-spin™ Total DNA Extraction Mini Kit (iNtRON BIOTECHNOLOGY, Gyeonggido, South Korea), following the manufacturer's protocol. A set of primers (listed in Table 2 [35]) used to amplify plantaricin genes was designed and PCR was performed using a 2X TOPsimple™ PreMIX-nTaq (Enzynomics, Daejeon, South Korea) and SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA), as previously described [35]. The PCR conditions were as follows: initial denaturation at 94 • C for 2 min, followed by 30 cycles of denaturation at 94 • C for 15 s, annealing at 60 • C for 30 s, and elongation at 72 • C for 1 min, and a final elongation step at 72 • C for 4 min. The PCR amplicons were subjected to 3.0% (w/v) agarose gel electrophoresis at 100 V for 40 min. The gel was visualized using the ChemiDoc™ MP Imaging System Universal hood III (Bio-Rad, Hercules, CA, USA) with Image Lab™ Software Version 5.2 (Bio-Rad).

Statistical analysis
The data of this study are presented as the mean ± standard deviation (SD) from at least three independent experiments with triplicate samples. For co-cultured assay, cultivation assay, and plate agar overlay assay, multiple groups were analyzed using Bonferroni's test for multiple comparisons of one-way analysis of variance (ANOVA) using GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA). p-values of <0.05, <0.01, and <0.001 were considered statistically significant.
Antimicrobial activity of APsulloc 331261 and APsulloc 331266 against S. aureus was further examined by agar overlay assay. Various populations (1 × 10 4 , 2 × 10 4 , 4 × 10 4 CFU) of APsulloc 331261 or APsulloc 331266 were spot inoculated on MRS agar plates, which were overlaid with TS agar containing S. aureus. We determined the growth inhibition of S. aureus by the measurement of zone of diameter inhibition (ZDI) and the wide of clear zone (R value). APsulloc 331261 or APsulloc 331266 spotted at 20.3 ± 2.1, 26.7 ± 3.1 and 28.7 ± 4.2 mm, respectively (Fig. 1C,D). Furthermore, R values for APsulloc 331261 or APsulloc 331266 against S. aureus ranged 6.8 ± 0.3-0.8 ± 1.3 mm or 5.7 ± 0.8-7.7 ± 1.4 mm, respectively, indicating the high inhibition capacity of APsulloc 331261 and APsulloc 331266 against S. aureus, but there was no dose dependent effect of APsulloc 331261 and APsulloc 331266 on R value. This might be due to dose dependent growth of APsulloc 331261 and APsulloc 331266 on the agar plate despite of its effect on dose dependent inhibition of the S. aureus growth. Collectively, agar overlay assay suggests that APsulloc 331261 and APsulloc 331266 exhibit strong inhibition (ZDI >20 mm, R value >6) against S. aureus. Taken all together, APsulloc 331261 and APsulloc 331266 have significant antibacterial effect against skin pathogenic S. aureus.

The growth of M. globosa and M. restricta was suppressed by APsulloc 331261 and APsulloc 331266
To examine antifungal activity, the viability of M. globosa and M. restricta co-cultured with APsulloc 331261 and APsulloc 331266 was evaluated in a transwell chamber. These experiments revealed that M. globosa and M. restricta growth was significantly decreased in the presence of APsulloc 331261 or APsulloc 331266 at ratio 1:0.1 and 1:1 (Fig. 4A,C). We also tested the effect of various concentrations (25, 50,  Plantaricin, a bacteroicin produced in L. plantarum, is known to have an antimicrobial effect [36]. To de- termine whether APsulloc 331261 and APsulloc 331266 express plantaricin genes, we evaluated ten genes, which are present in the pln locus and are responsible for the synthesis of plantaricins. PCR analysis showed that plnA, plnH, plnG, plnEF, plnJ, plnK, and plnN genes exist in APsulloc 331261 and APsulloc 331266, and that plnS, plnW, and plnNC8αβ genes do not exist (Fig. 4E,F). The plantaricin genes such as plantaricin S and plantaricin W are rarely presented in the L. plantarum strains [36]. Taken together, Genes plnA, plnH, plnG, plnEF, plnJ, plnK, and plnN were found in APsulloc 331261 and APsulloc 331266, which may render its antimicrobial activity against pathogenic skin microbes.

Discussion
The usage of probiotics to promote gut and immune health has recently grown, and researchers are inter- After incubation at 37 • C for 48 h, the cell viabilities were determined at OD 600 . (C,D) ZDI and R value of plate agar overlay assay with C. albicans were determined. All data represent the mean ± standard deviation (SD) from at least three independent experiments with triple samples. Significant differences: * p < 0.05, * * p < 0.01, * * * p < 0.001 vs. (-), # p < 0.05, ## p < 0.01 vs. 1 × 10 4 CFU of initial inoculation. ested in the discovery of a variety of probiotic strains and mechanisms how probiotic microorganisms interact with the body [37]. Probiotics play an potential alternative to antibiotics in the treatment of inflammatory bowel disease (IBD) [38] as well as lower the incidence of several atopic conditions [39,40]. Among the commercially available probiotic strains, LAB from various natural sources are being isolated and identified to explore new alternative probiotic strains [41]. Probiotic Lactoplantibacillus species can be considered to be candidates for alternatives to antibiotics since their various mechanisms of growth inhibition against pathogenic bacteria have been extensively documented [42][43][44]. Lactiplantibacillus species isolated from various sources have an excellent antibacterial spectrum against pathogenic bacteria [5,45,46]. The antagonistic activity of Lactiplantibacillus species has mostly been attributed to the production of lactic acid. The acidification of cytoplasm of cell by the undissociated lactic acid that passes through the bacterial membrane and dissociates inside the cell results in failure of proton motive forces [47]. Besides organic acids, hydrogen peroxides, bacteriocins, and short-chain fatty acids (such as acetic, propionic, and butyric acids) are antimicrobial compounds produced by Lactiplantibacillus species, and the compounds lead to the change of the gut redox potential and the restriction of the nutrient supply to the pathogens [48]. In particular, L. plantarum synthesize a variety of antimicrobial plantaricins to inhibit the growth of competing bacteria. Plantaricin A, which plnA is encoded, acts as an extracellular signal that induces transcription of pln genes as well as an antimicrobial peptides [49]. Even though plantaricin E, F, J and K, encoded by plnE, plnF, plnJ and plnK, respectively, have an anti-microbial effect individually, two peptides are at least 1000 times more effective when cognate peptides such as PlnE and PlnF, or, PlnJ and PlnK, are combined [50]. Moreover, antifungal activity of plantaricin peptides PlnE, -F, -J, and -K is presented against pathogenic yeast, Candida albicans [51]. Plantaricin EF and JK form holes in the membrane of the target cell and cause the death by electronical potential and pH gradient to collapse [52,53]. However, plantaricin EF and JK have different receptor [54]. plnH and plnG is related to ABC transport, which secrete and process pre-plantaricin E, F, J, and K to reveal the antimicrobial activity [52]. Yolanda et al. [55] classified the L. plantarum strains into seven plantaritypes, depending on the presence of the bacteriocin gene and the type of regulatory system. Both APsulloc 331261 and APsulloc 331266 do not have plnNC8, but plnA does exist, suggesting that they belong to group 1 and 2 of plantaritypes and have common characteristics of plnABCD regulation systems. Thus, the presence of plantaricin genes plnA, plnH, plnG, plnEF, plnJ, plnK, and plnN in APsulloc 331261 and APsulloc 331266 might contribute to the antimicrobial activity of APsulloc 331261 and APsulloc 331266 against skin pathogens. However, we only identified plantaricin genes, not their expression or activity. For better understanding of the mechanism of antimicrobial activity of APsulloc 331261 and APsulloc 331266, further studies are needed to confirm the plantaricin expression/activity and their antimicrobial activity against skin pathogenic microbiota.
Probiotics and prebiotics have been widely considered for the treatment and prevention of infectious diseases because of the various mechanisms by sharing genes and metabolites, helping other microbiota, and directly interacting epithelial and immune cells [56]. In this regard, several studies have investigated the effects of probiotic and prebiotics use on microbiota of other parts of human body. Therefore, probiotics and prebiotics are promising in improving skin health by modulating the cutaneous microbiota [57]. The gastrointestinal microbiota can communicate with the skin through modulation of systemic immune responses and maintain skin homeostasis [58]. In fact, beneficial effects of probiotics following topical or oral administration have been reported in skin diseases, such as acne vulgaris [27,59,60], atopic dermatitis [61], candida vaginitis [62], and dandruff [63]. The use of probiotics in cosmetics or nutraceuticals have been recently expanded to rebalance the microbiome for skin by inhibiting the growth of harmful pathogens while promoting the colonization of beneficial bacteria among the resident microorganisms. Thus, topical application or ingestion of APsulloc 331261 and APsulloc 331266 could improve skin health by inhibiting skin pathogens, ameliorating intestinal beneficial bacteria [30] and inducing macrophage polarisation [31]. Although we should carefully draw conclusion obtained from in vitro studies, our results support the hypothesis that probiotic APsulloc 331261 and APsulloc 331266 may ameliorate microbial profile and homoeostasis in the skin. Further studies are guaranteed to investigate the effectiveness of APsulloc 331261 and APsulloc 331266 as novel antibiotics or alternative therapeutics for treating skin infection.

Conclusions
In summary, the present study demonstrated that APsulloc 331261 and APsulloc 331266 isolated from green tea (Camellia sinensis) exhibited the inhibitory activity against five skin pathogenic strains (S. aureus, C. acnes, C. albicans, M. globosa and M. restricta) related to skin diseases. Antimicrobial effect of APsulloc 331261 and APsulloc 331266 was validated by indirect co-culture assay, CM culture assay and agar overlay assay with five pathogenic strains. These results indicate that novel APsulloc 331261 and APsulloc 331266 could serve as a potential alternative to antibiotics against infectious diseases. Further studies are guaranteed to explore the effectiveness of APsulloc 331261 and APsulloc 331266 administrated by topically or orally as alternative therapeutics in combating skin infection.

Author contributions
MC, BJK and JN designed the research and wrote the paper. JN, JOL and SYK performed the experiments and statistical analyses. YK, EL, DC, JR and WK prepared figures. All authors analysed and interpreted the data, revised the manuscript, and approved the final draft.

Ethics approval and consent to participate
Not applicable.

Acknowledgment
Thanks to all the peer reviewers for their opinions and suggestions.

Funding
This research was funded by the AMOREPA-CIFIC R&D center (R21S200001).

Conflict of interest
The authors declare no conflict of interest.