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Immunomodulatory effects of seven viable and sonicated Lactobacillus spp. and anti-bacterial activities of L. rhamnosus and L. helvetilus
Korean J. Microbiol. 2019;55(4):392-399
Published online December 31, 2019
© 2019 The Microbiological Society of Korea.

Yae-Jin Choi, Hyun-Jung Lim, and Hea-Soon Shin*

College of Pharmacy, Duksung Women’s University, Seoul 01369, Republic of Korea
Correspondence to: *E-mail: hsshin@duksung.ac.kr;
Tel.: +82-2-901-8398; Fax: +82-2-901-8386
Received October 16, 2019; Revised November 1, 2019; Accepted November 4, 2019.
Abstract

Usage of probiotic lactic acid bacteria (LAB) has recently attracted more attention due to increased interests in human health. They are also known to possess immunomodulatory effects by promoting cytokine secretion and thus modulate inflammatory responses. This study has aimed to evaluate the immunomodulatory effects and antimicrobial activities of 7 Lactobacillus strains that had been obtained from Korean Collection for Type Culture. The RAW 264.7 cell line was used as a macrophage model to investigate into assess the non- specific effects of seven Lactobacillus strains on the production of NO as well as the cytokines TNF-α and IL-1β. The minimum inhibitory concentrations of several antibiotics for the Lactobacillus strains and the anti-bacterial activities of viable and sonicated cells, from three select Lactobacillus spp., were measured on the growth of pathogenic bacteria. The sonicated LAB also stimulated IL-1β and TNF-α production compared with the RAW cell only. In addition, sonicated L. rhamnosus and L. helvetilus were found to have higher anti-bacterial activity than the viable LAB in this study. These results suggest that sonicated LAB could be used to modulate immune responses with strain- dependent.

Keywords : Lactobacillus species, anti-bacterial activity, immunomodulatory effect
Body

Lactic acid bacteria (LAB), such as Streptococcus spp., Lactobacillus spp., Lactococcus spp., Bifidobacterium spp., and Enterococcus spp. These bacteria decompose carbohydrates to produce lactic acid and are classified as facultative anaerobe bacteria in terms of oxygen conditions for bacterial growth. Previous studies have shown that LAB strains are beneficial in the prevention and treatment of inflammatory bowel disease since they aid lactose absorption, improve immune function, have anti-cancer effects, synthesize vitamins, control serum cholesterol levels, and inhibit growth of harmful bacteria (Lee et al., 2016b).

Besides, advantages of Lactobacillus spp., such as L. acidophilus and L. casei, also been recently studied. Lactobacillus spp. are the most commonly used probiotics along with Bifidobacterium. They help to equilibrate the intestinal microbiota, have antimicrobial and enzymatic activities, and produce metabolites useful for the host, thereby promoting host health. Lactobacillus spp. (Mottet and Michetti, 2005), being normal intestinal flora, normalize gastrointestinal diastalsis and promote secretion such as amylase, cellulose, lipase, and protease, thereby promoting digestion and food absorption. Since Lactobacillus spp. are more acid-tolerant than other LAB species and are capable of proliferating to some extent in aerobic conditions, enabling them are likely to survive and pass through the stomach to reach the intestines where they can proliferate easily.

LAB strains interact with macrophages and T cells to secrete a variety of cytokines with beneficial effects on both immune and non-immune cells. In addition, they are known to enhance the abilities of macrophages to recognize and remove harmful intestinal bacteria, promote secretion of immune-related substances for immunomodulatory effects (Li et al., 2009; Sim et al., 2016). Macrophages are stimulated by cytokines such as lipopolysaccharide (LPS) from Gram-negative bacteria in vitro and in vivo, and activated microphages are responsible for cell-mediated immunity. Activated macrophages not only kill targeted cells but also secrete cytotoxic substances such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, nitric oxide (NO), and cytolytic protease. It has been reported that NO produced in this manner can inhibit growth of bacteria, viruses, and tumor cells.

NO is produced by inflammatory cells by activated inducible nitric oxide synthase (iNOS). LPS, which is an external stimulator, is composed of lipid A and various polysaccharides, and even a small amount of LPS induces an immune response. LPS functions by binding to LPS-binding protein (LBP) and CD14 receptor and then activates toll-like receptor (TLR) 4, thereby producing interferon (IFN)-β. Then, IFN-β binds to its cell receptor in order to phosphorylate signal transducer and activator of transcription (STAT1) protein and induce production of iNOS (Yun et al., 2007).

Macrophages also regulate immune function by increasing production of mediators such as TNF-α and IL-1β. These mediators play important roles in regulating erythropoiesis, lymphocyte production, platelet production, and homeostasis (Chuang et al., 2007). In the case of gram-positive bacteria, lipoteichoic acid (LTA), a component of cell walls with fatty acids and various polysaccharides, is substance that causes infections. LTA and LPS bind to binding proteins or carrier proteins to activate TLR and induce expression of inflammatory cytokines such as TNF-α and IL-1β (Hong et al., 2016). Infection with pathogenic bacteria leads to activation of immune cells, resulting in excessive secretion of inflammatory factors.

Since LAB produce bacteriocins with anti-bacterial activity as well as large amounts of lactic acid and acetic acid as active metabolites, LAB strains can prevent metabolic disease when used as probiotics by maintaining the intestinal microbiota equilibrium and promoting health (Yang et al., 2012).

Sonicated LAB have the advantages of a longer shelf life, easier storage, and easier transportation. In addition, LAB strains show immunostimulatory effects by promoting proliferation of immune cells, thereby selectively removing harmful intestinal bacteria and foreign substances in the intestines and promoting secretion of various immunoproteins and enzymes (Zelaya et al., 2015).

Oral administration of heated LAB could also be a convenient treatment for patients with perennial allergic rhinitis (Peng and Hsu, 2005). In addition, it has been reported by literature that antibiotics and anti-tuberculosis drugs administered to treat chronic diseases may destroy normal intestinal flora and cause intestinal diseases such as malabsorption and indigestion. In such cases, probiotics can be administered in combination with therapeutic antibiotics (Lee et al., 2011).

In this study, we investigated NO, IL-1β, and TNF-α production using RAW 264.7 cells as an in vitro murine macrophage model to determine the immunostimulatory effects of seven Lactobacillus strains. We measured the minimum inhibitory concentrations (MICs) of antibiotics against Lactobacillus strains and investigated the possibility of using LAB as probiotics for chronic disease that require antibiotics for a long period. In addition, growth inhibition experiments were conducted to investigate into the antimicrobial effects of three strains of sonicated Lactobacillus spp., selected for its efficacy, against methicillin-resistant Staphylococcus aureus ATCC 33592 and vancomycin-resistant Enterococcus faecalis ATCC 52199.

Materials and Methods

Materials

Dulbecco’s modified eagle’s medium (DMEM), FBS, penicillin (10,000 U/ml)/streptomycin (10,000 U/ml), and LPS (Escherichia coli, 0127:B8 Westphal type) were purchased from Sigma. The antibiotics used to measure the MICs were ciprofloxacin (Ildong), gentamicin (Kukje), lincomycin (Yuyu), cycloserine (Donga), meropenem (Yuhan), amoxicillin/clavulanic acid (IlSung), and cefazolin (Yuhan). Gifu anaerobic medium (GAM) broth (Difco) was used as the preincubated media for LAB, whereas brain heart infussion (BHI) broth (Difco) was used as the preincubated media for MRSA and VRE.

Strains

RAW 264.7 cells were purchased from the Korean Cell Line Bank (KCLB) and stored in a nitrogen tank. Cells used for the experiment were incubated at 37°C in a CO2 incubator using DMEM with 10% fetal bovine serum (FBS), 1% streptomycin (10,000 U/ml), and penicillin (10,000 U/ml) added. RAW 264.7 cells were put in a sterile glass-slide chamber (Nunc) at a density of 1 × 103 cells/well.

Seven Lactobacillus spp.: Lactobacillus gasseri KCTC3144, Lactobacillusparacasei KCTC13090, Lactobacillus delbrueckii ssp bulgaricus KCTC3635, Lactobacillus rhamnosus KCTC13088, Lactobacillus reuteri KCTC3594, Lactobacillus helveticus KCTC15060, and Lactobacillus salivarius salicinus KCTC3156 were purchased from the Korean Collection for Type Culture (KCTC).

The LAB strains used in this experiment are listed in Table 1. After incubation for 24 h, the LAB were centrifuged to obtain the supernatants, which were filtered and sterilized using a syringe filter. The pathogenic bacteria used for the MIC test were methicillin-resistant Staphylococcus aureus (MRSA) American Type Culture Collection (ATCC) 33592 and vancomycin-resistant Enterococcus faecalis (VRE) ATCC 52199.

Minimal inhibitory concentrations of several antibiotics for Lactobacillus strains

StrainsMICs (µg/ml)

CIPGENLINCYCSMEMAMCCFZ
Lactobacillus gasseri KCTC3144<12.54<0.5>1006.25>100>100
Lactobacillus paracasei KCTC13090<6.25ND50>1001.6>100>100
Lactobacillus delbrueckii ssp bulgaricus KCTC3635<32<4>100>1001.6>100>100
Lactobacillus rhamnosus KCTC13088<6.258<0.5>1001.6>100>100
Lactobacillus reuteri KCTC3594<321.6>100>10012.5>100>100
Lactobacillus helveticus KCTC15060<12.5ND<0.5>1006.25>100>100
Lactobacillus salivarius salicinus KCTC3156<32<0.4ND>1006.250.4>100

CIP, ciprofloxacin; GEN, gentamicin; LIN, lincomycin; CYCS, cycloserine; MEM, meropenem; AMC, amoxicillin/clavulanic acid; CFZ, cefazolin; ND, Not determined


Nitric oxide (NO) assay

LPS (10 ng/ml), seven Lactobacillus strains, RAW 264.7 cell line, and Griess reagent (stock-I: 0.2% naphthalene diamine HCl, stock-II: 2% sulfanilamide in 5% H2PO4) were used as materials. A total of 96 wells per group were used, and 200 µl of cells (1 × l06 cells/ml) was added to each well. NO produced by macrophages remains present for 6~8 sec, after which it is spontaneously oxidized into NO2 and NO3. Therefore, to precisely quantify the reactive nitrogen intermediate (RNI) produced, NO3 should be converted into a reduced element. Since most NO accumulates as NO2, indirect quantification was performed (Anukam, 2009). The plates were incubated overnight, and 100 µl from the surface of each well was transferred into a new plate with the equivalent amount of Griess reagent. The new plate was incubated for 10 min at room temperature and measured by an ELISA reader at 540 nm.

TNF-α and IL-1β assay

The culture broth of RAW 264.7 cells treated with viable and sonicated LAB was harvested, and 100 μl of the harvested culture broth and standard were added to each well and incubated at room temperature for 90 min after adding 50 µl of biotin conjugate. The mouse immunoassay kit (BioSource) was used to quantify TNF-α and IL-1β secreted in the culture broth after incubation. Wells were coated overnight at 4°C with 100 µl of 1 μg/ml of purified anti-mouse TNF-α and IL-1β antibodies in 0.1 M sodium bicarbonate buffer (pH 8.2). Wells were incubated with 200 µl of 3% bovine serum albumin in 0.1 M PBS (pH 7.2) containing 0.2% Tween 20 at room temperature for 1 h to block non-specific protein binding. Then, after washing eight times, bound peroxidase conjugate was detected by adding, per well, 100 µl of substrate solution consisting of 0.1 mg/ml of tetramethylbenzidine and 100 ml of 1% H202 in 25 ml of 0.1 M citric phosphate buffer (pH 5.5). The pro-inflammatory cytokines TNF-α and IL-1β were confirmed through ELISA at 450 nm.

Minimum inhibitory concentrations test

The minimum inhibitory concentration was measured by the solid medium dilution method. The MICs of several antibiotics against Lactobacillus spp. strains were tested. The antibiotics tested were amoxicillin/clavulanic acid, ciprofloxacin, gentamicin, lincomycin, cycloserine, meropenem, and cefazolin. The concentration of each antibiotic was diluted by a factor of two in a stepwise manner, and each antibiotic was added to nutrient broth agar and solidified in a plate. The test strains were transplanted in Mueller-Hinton agar and cultured in an incubator at 37°C for 24 h. The numbers of cells were counted per 0.5 McFarland standard and then adjusted to a final bacterial concentration of 1 × 104 CFU/ml for use as bacterial solutions for the MIC tests. Then, 50 µl of each prepared bacterial solution was smeared onto a plate containing each antibiotic and incubated at 37°C for 48 h. Turbidity of the test broth was measured in terms of optical density at various dilutions. When optical density values for each dilution of the LAB test broth and the pure bacterial culture medium were nearly identical, the minimum inhibitory concentration of the LAB test broth was taken as the MIC.

Disc diffusion assay

We tested the anti-bacterial activity of LAB by utilizing the disc diffusion method. LAB strains were inoculated into MRS broth and incubated at 37°C for 48 h, after which the supernatant was recovered by centrifugation at 4,000 rpm for 10 min. The recovered supernatant was filtered using a 0.2-μm syringe filter. The antimicrobial activity of the concentrated culture supernatant was assessed by the disc diffusion assay. Pathogenic intestinal bacteria were cultured in an aerobic environment, and beneficial intestinal bacteria were cultured in an anaerobic chamber for 24 h. The antimicrobial activity was assessed by smearing the bacterial solution with presumed anti-bacterial activity on the agar culture media after preparing each of the bacterial solutions at a concentration of 1.5 × 108 CFU/ml in Mueller-Hinton agar. Each 6.35 mm disc was infused with 60 μl of each sample for assessment of antimicrobial activity and then placed in culture medium and incubated for 18~24 h at 37°C in an incubator, after which the diameter of the growth inhibition zone was measured. The pathogens used to evaluate antimicrobial activity were MRSA (methicillin-resistant Staphylococcus aureus) ATCC 33592 and VRE (vancomycin-resistant Enterococcus faecalis) ATCC 52199. MICs and disc diffusion assay were performed based on the guidelines of the Clinical Laboratory Standards Institute with some modifications (Clinical Laboratory Standards Institute, 2012).

Results and Discussion

Lactic Acid Bacteria (LAB) can stabilize gut microbiota, inhibit intestinal colonization of harmful bacteria, enhance immunity, colonize intestinal cells, and exhibit antimicrobial activity to maintain the gut microbiota equilibrium. Among them, Lactobacillus spp., classified as a facultative anaerobic bacillus, is the first type of LAB to be utilized in the form of fermented milk to maintain the intestinal flora equilibrium (Schillinger and Lücke, 1989; Hong et al., 2016). A study by Shin showed that L. casei and L. acidophilus have high acid resistance and that L. acidophilus survives at pH 2~3 for more than 30 min (Shin et al., 2015). LAB have the advantage of being safe to use in people with a weak immune system. In addition, elevation of cytokine production by macrophages is known to enhance immunity.

NO production ability

Since NO is an important mediator of macrophages, the effect of LAB on NO production was assessed by Griess assay. The effects of seven Lactobacillus strains on NO production in RAW 264.7 cells were examined, and almost no amount of NO was released in the group treated with only medium, as shown in Fig. 1. The group in which RAW 264.7 cells were treated with LPS was used as the positive control for macrophage activity. In the group treated with the supernatants of seven Lactobacillus strains, NO production was relatively higher than in the control group. In particular, NO production was highest in the groups treated with L. rhamnosus, L. helveticus, and L. paracasei.

Fig. 1.

NO production from RAW 264.7 cells by treatment of seven Lactobacillus spp. Cells, supernatant of media; Control, LPS (100 ng/ml); significant differences (** P < 0.01) between untreated cells.


When the seven Lactobacillus strains were added to the culture media of differentiated RAW 264.7 cells, NO production was increased, as shown in Fig. 1. When RAW 264.7 cells were treated with a 22-fold diluted solution of each of the seven strains of Lactobacillus, the amount of NO produced was significantly higher than in the group treated only with LPS (10 ng/ml) as the positive control (Fig. 1). Substances such as IFN-β and LPS are known to induce NO2 production in culture media through macrophage activation (Lee et al., 2016a). These seven strains of Lactobacillus also activated macrophages and thus promoted production of NO.

Quantification of TNF-α and IL-1β

LAB are known to regulate immunomodulatory effect in the body by promoting secretion of cytokines and are involved in inflammatory responses. Macrophages also regulate immune function by increasing production of mediators such as TNF-α and IL-1β (Sim et al., 2016). To investigate the effects of viable and sonicated Lactobacillus spp. on the production of the pro-inflammatory cytokines TNF-α and IL-1β, RAW 264.7 cells were cultured with seven strains of Lactobacillus in the presence or absence of LPS, and the results are shown in Lee et al., 2016aFigs. 2 and 3.

Fig. 2.

TNF-α production from RAW 264.7 cells with LPS by treatment of seven Lactobacillus spp. Cells, supernatant of media; Control, LPS (0.1 pg/ml); significant differences (* P < 0.05) between untreated cell.


Fig. 3.

IL-1β production from RAW 264.7 cells with LPS by treatment of seven Lactobacillus spp. Cells, supernatant of media; Control, LPS (0.1 pg/ml); significant differences (* P < 0.05) between untreated cells.


In the present study, the amounts of IL-1β produced were lowest in the LPS-untreated group, and IL-1β levels increased in a dose-dependent manner in groups treated with the 22-fold dilutions of the Lactobacillus strains, as compared to the group treated only with LPS (10 ng/ml) as the positive control. The amounts of TNF-α and IL-1β produced were also higher in the groups treated with L. rhamnosus, L. helveticus, L. paracasei, and L. gasseri at 22-fold dilution than in the group treated only with LPS. In addition, the amounts of IL-1β produced were significantly higher in the group treated with both LAB and LPS than in the group treated only with LAB (Fig. 3). All seven Lactobacillus strains generally up-regulated production of TNF-α and IL-1β. These results seem to indicate that Lactobacillus spp. stimulated macrophages to produce TNF-α and IL-1β, which will lead to enhanced immune function (Yun et al., 2006).

Macrophages are responsible for initial and non-specific immune responses, and substances such as LPS and IFN-α induce specific activity. In the present study, all seven Lactobacillus strains induced the production of TNF-α and IL-1β and macrophage activation. To determine whether or not Lactobacillus strains have a direct effect on cytokine production in unstimulated and LPS-stimulated groups, activation of both TNF-α and IL-1β was measured using the macrophage cell line. In addition, much greater production of both TNF-α and IL-1β was observed with sonicated Lactobacillus spp. compared to controls.

Measurement of MICs

The minimum inhibitory concentrations were measured by the solid medium dilution method based on the method of NCCLS (National Committee for Clinical Laboratory Standards) using seven kinds of antibiotics. The biomass was inoculated into plate media by the solid medium dilution method. The antibiotics used were amoxicillin/clavulanic acid, ciprofloxacin, cycloserine, gentamicin, lincomycin, meropenem, and cefazolin. LAB solutions with concentrations adjusted to 104 CFU/ml were used for the MIC test, and the results are shown in Table 1. According to these results, L. paracasei and L. helveticus showed high resistance (not detected) to gentamicin compared with other Lactobacillus strains. All tested strains showed high resistance to amoxicillin/clavulanic acid (except L. salivarius), cycloserine, and cefazolin (> 100 µg/ml), and the MIC values of ciprofloxacin for the seven Lactobacillus strains were 6.25~32 µg/ml. All tested Lactobacillus strains were sensitive to meropenem. However, L. paracasei, L. delbreuckii, L. reuteri, and L. salivarius showed high resistance to lincomycin.

Anti-bacterial activities of selected LAB strains

The antimicrobial activities of L. rhamnosus, L. helveticus, and L. paracasei, selected by screening in the previous assay, were assessed. The concentrated culture supernatants of the strains were exposed to methicillin-resistant Staphylococcus aureus ATCC 33592 and vancomycin-resistant Enterococcus faecalis ATCC 52199, which are multiple drug-resistant bacteria, and incubated for 24 h to assess anti-bacterial activity based on the formation of clear zones around the disc.

Recently, L. sakei and L. casei, have been reported to have similar anti-bacterial activities against strains such as B. cereus and E. coli (Jorjão et al., 2018). Most prior studies have focused on the immunomodulatory effects of viable cells of LAB, whereas the immunomodulatory effects of sonicated cell of LAB have been investigated in this study. Three strains of sonicated Lactobacillus spp. were selected to measure antimicrobial activities (Table 2). As a control, media cultured without Lactobacillus spp. were used, and no clear zones were observed. Three selected sonicated Lactobacillus spp. showed clear zones with a diameter of up to 12.5 mm against E. coli in most cases as well as clear zones with a diameter of at least 10 mm against MRSA ATCC 33592. Sonicated L. rhamnosus, L. helvetilus, and L. paracasei exhibited clear zones with a diameter of at least 10 mm against VRE ATCC 52199. These findings are consistent with previous studies reported that LAB strains have anti-bacterial activities against pathogenic bacteria such as S. aureus, E. coli, Pseudomonas aeruginosa, and Salmonella typhi (Choi et al., 2014).

Diameter of growth inhibition zone by viable and sonicated Lactobacillus spp.

Lactic acid bacteriaGrowth inhibition zone (mm)

S. aureusaE. faecalisbE. coli
Viable - L. rhamnosus KCTC1308813 ± 0.514 ± 0.25 16 ± 1.0
Viable - L. helvetilus KCTC1506011 ± 1.013 ± 0.5 19 ± 0.5
Viable - L. paracasei KCTC1309011 ± 0.2512 ± 0.5 13 ± 0.75
Sonicated - L. rhamnosus KCTC1308814 ± 0.2515 ± 0.5 18 ± 0.25
Sonicated - L. helvetilus KCTC1506015 ± 0.2516 ± 0.75 19 ± 0.5
Sonicated - L. paracaei KCTC1309010 ± 1.011 ± 0.5 12 ± 0.25

methicillin-resistant Staphylococcus aureus ATCC 33592

vancomycin-resistant Enterococcus faecalis ATCC 52199, Values represent the mean ± SD.


Lactobacillus spp. are known to inhibit the growth of pathogenic microorganisms through lactic acid, bacteriocin (an active metabolite of LAB), and their relatively good ability to adhere to intestinal epithelial cells (Schillinger and Lücke, 1989). Probiotic LAB survive to reach the intestinal tract by altering the pH through production of lactic acid, acetic acid, and organic acid as well as by promoting intestinal epithelial cell adhesion and specific cytokine production. Sonicated LAB, which are not to proliferate, produce metabolites such as organic acids and bacteriocin.

The results of this study suggest that sonicated LAB could be used to modulate immune responses with strain-dependent. Sonicated LAB have the advantages of a longer shelf life, easier storage, and easier transportation. Oral administration of sonicated LAB may conveniently be used for immunoprophylaxis. In addition, the first defense mechanism of probiotics and adherence to the gut mucosal membrane is inhibition of pathogenic infection. The approaches employed here could be useful in further characterization of the regulatory effects of LAB on systemic immunity.

적 요

LactobacillusBifidobacterium과 함께 가장 많이 이용되고 있는 probiotics로서 장내 미생물 균형에 도움을 주고 항균 활성과 면역 조절효과를 가지면서 유용한 대사물을 생산하는 기능이 보고되어 있다. Lactobacillus는 다른 lactic acid bacteria와 비교하여 내산성이 높고, 산소조건에서도 증식이 가능해서 위를 통과하여 장까지 보내지는 양이 비교적 더 많거나 증식도 쉽게 되기 때문이다. 본 연구에서는 7종의 Lactobacillus의 macrophage 활성화 여부로 면역 증진 효과를 확인하고자 숙주 면역 기능을 갖는 NO 생성 정도와 macrophage의 mediator인 TNF-α, IL-1β 생성 정도를 조사하였다. 항생제의 최소 성장 억제 농도(minimum inhibitory concentration)를 측정하고 면역 증진 효과를 나타낸 Lactobacillus 3종(L. rhamnosus, L. helvetilus, L. paracasei)을 선별하여 다제내성 병원균인 methicillin- resistant Staphylococcus aureus (MRSA)와 vancomycin-resistant Enterococcus faecalis (VRE)를 억제하는 효과를 나타내는지 알아보기 위해 성장 억제 실험을 수행하였다. 비교된 결과로서 초음파 처리된 L. rhamnosusL. helvetilus의 항균활성이 생균의 경우보다 높은 결과를 나타내었다. 이는 lactic acid bacteria의 성장이 정지된 채 bacteriocin이나 유기산 등 대사물을 생산하기 때문인 것으로 사료된다.

Acknowledgements

This research was supported by Basic Science Research Program through the NRF funded by the Ministry of Education 2017R1D1A1B03031273.

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