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Section 2. Biotechnology
DOI: http://dx.doi.org/10.20534/ESR-16-11.12-16-20
Sarmurzina Zinigul,
Head of Laboratory of Microbiology in RSE "Republican Collection of Microorganisms" SC MES RK (Astana, Kazakhstan), Candidate of biological science RSE "Republican Collection of Microorganisms" SC MES RK, Astana, Kazakhstan Bissenova Gulmira, Senior Researcher of Laboratory of Microbiology in RSE "Republican Collection of Microorganisms" SC MES RK (Astana, Kazakhstan), Candidate of agricultural science RSE "Republican Collection of Microorganisms" SC MES RK, Astana, Kazakhstan Kunsulu ZaKarya, Deputy General Director for Science in RSE "Republican Collection of Microorganisms" SC MES RK (Astana, Kazakhstan), Doctor of biological sciences RSE "Republican Collection of Microorganisms" SC MES RK, Astana, Kazakhstan
Dospaeva Raikhan, Junior researcher of Laboratory of Microbiology in RSE "Republican Collection of Microorganisms" SC MES RK (Astana, Kazakhstan), Bachelor degree in Biotechnology RSE "Republican Collection of Microorganisms" SC MES RK, Astana, Kazakhstan Abzhalelov Akhan,
Director General in RSE "Republican Collection of Microorganisms" SC MES RK (Astana, Kazakhstan), Doctor of biological sciences RSE "Republican Collection of Microorganisms" SC MES RK, Astana, Kazakhstan E-mail: rcm-info@mail.ru
Screening of lactic acid bacteria for antagonism toward pathogens and biofilm-forming activity
Abstract: Probiotic preparations based on lactic acid bacteria are used widely in clinical practice. Probiotic bacteria must have a set of properties that allow them to compete with pathogens and opportunistic pathogens in the gut. These characteristics are antagonism, bacteriocin-producing activity, the ability for adhesion, resistance to hydrochloric acid and bile, and safety in use. Twenty-five strains of lactic acid bacteria (LAB) from four genera were obtained from the Republican Collection of Microorganisms: Lactobacillus (19 strains), Lactococcus (3), Pediococcus (2), and Leuconostoc (l). We screened these LAB for antagonism toward pathogenic and biofilm forming activity. Antagonism was determined by the agar diffusion method against Escherichia coli, Staphylococcus aureus, Candida albicans, and Serratia marcescens. Determination ofbiofilm-forming activity was carried out by serial dilutions ofbiofilms formed in vitro on two materials, cover glass and polystyrene plate. The most antagonistic activity was showed against S. aureus and E. coli. As a result of the investigations were selected strains: Lactobacillus casei 3, which showed antagonism toward conditionally pathogenic microorganisms; L.plantarum 8RA-3 pl+ and L. casei r, which have high biofilm forming ability.
Keywords: Lactic acid bacteria, Antagonism; Biofilm formation.
Introduction
One of the commonest current directions ofmicrobiology is the search for new strains oflactic acid bacteria (LAB) to create probiotic products. One of the major components of starter cultures for such drugs is typically Lactobacillus bacteria. Naturally introduced probi-
otic cultures have a positive impact on the physiological, biochemical and immune response of the host organism via stabilization and optimization of its normal microflora function. Microorganisms of the genus Lactobacillus are widespread in nature, and some species are the most important representatives of the microbiota of the human
body [1; 2]. Because they produce organic acids, hydrogen peroxide and bacteriocins, many strains oflactobacilli show antagonistic activity toward pathogenic and conditionally pathogenic microorganisms [3; 4; 5]. In aggregate, these characteristics are the main mechanisms of action of Lactobacillus to inhibit the growth and reproduction of pathogenic and conditionally pathogenic microorganisms, in restoring microbiocenosis in the intestinal biotope, in strengthening the barrier function of intestinal epithelial cells, and in modulation of the immune response [6; 7; 8; 9].
In medical practice, research into and prevention of pellicle formation by pathogenic and opportunistic pathogenic microorganisms is particularly important, as this contributes to chronic infection and weakens the effectiveness of treatment [10]. Nosocomial infections (infections acquired in hospital) are the fourth leading cause of death in the United States, and about 65% of these infections are caused by biofilms on implanted medical devices such as intravenous catheters [11]. Biofilms also show increased resistance to antibiotics [12]. The ability to form biofilms is an important property for both pathogenic bacteria and for industrially significant bacteria that are used in various processes, such as fermentation and/or preservation of food and feed [13]. Use of LAB and their metabolites is the most common and popular method of natural protection; protective biofilm agents may be formed by LAB. Current hypotheses concerning Lactobacillus biofilms protecting against pathogenic bacteria involve the production of antimicrobial metabolites or inhibitory extracellular polymeric substances that surround the pathogenic bacteria, modulating Lactobacillus-pathogen interfaces [14; 15; 16; 17].
The aim of this work was screening of LAB active cultures with high antagonistic and biofilm forming activity.
Materials and methods
This research used 25 strains of LAB from the Republic Collection of Microorganisms (RCM), Kazakhstan. Twenty-one strains were from the culture collection (Lactobacillus casei r B-RKM 0004, L. casei 3 B-RKM 0008, L. brevis 3-9 B-RKM 0010, L. fermentum 90T C 4-pl B-RKM 0014, L. plantarum 8RA 3-pl B-RKM 0015, L. plantarum pl 38 2/T B-RKM 0017, L. fermentum ATCC 9338 B-RKM 0018, L. casei L B-RKM 0027, L. delbrueckii subsp. lactis CT-1 r B-RKM 0044, L. fermentum 136 B-RKM 0103, L. plantarum 2 B-RKM 0152, L. fermentum 96 B-RKM 0155, L. fermentum B-RKM 0203, L. casei BI 005 B-RKM 0208, L. brevis L5 B-RKM 0347, L. brevis L9 RKM 0348, L. sakei 2 a B-RKM 0640, L. sakei 7a B-RKM 0636, Pediococcus pentosaceus 8a B-RKM 0635, Lactococ-cus (Lc.) garvieae 10 a B-RKM 0639, L. sakei 24 a B-RKM 0559). Also in this study were study four strains of LAB from the working collection: P. pentosaceus 1a, Leuconostoc (Ln.) garlicium 3 a, Lc. lactis 14a, and Lc. lactis 17a.
Determination of antagonistic activity of LAB
Antagonistic activity of LAB was determined by the agar diffusion method [18] with test strains Escherichia coli ATCC 25922 B-RKM 0447, Staphylococcus aureus 209 P B-RKM 0057, Candida albicans ATCC 885-653 Y-RKM 0475, and Serratia (Ser.) marcescens 221F B-RKM 0059, from the of RCM. E. coli ATCC
25922, S. aureus 209 P, and Ser. marcescens 221F were incubated overnight in nutrient broth (HiMedia, Mumbai, India) at 37 °C, and C. albicans ATCC 885-653 in Sabouraud dextrose broth (SDB, HiMedia) at 37 °C. A test-culture was a suspension of cells at 10 9/ml applied to the surface of a Petri dish containing meat-peptone agar (Nutrient agar (HiMedia) with Meat extract powder (Accumix, Belgium)). After this, a 5-mm-diameter hole was cut in the plate with a drill and filled with cultures of LAB (0.1 ml). Incubation was carried out at 37 °C. Zones of inhibition were measured after 48 h and are presented with the diameter of the hole subtracted. Antagonistic activity was evaluated by the lack of growth of the indicator strain around the cultures of the LAB strain.
Determination of biofilm-forming activity of LAB
To determine the biofilm forming activity of LAB, we used a serial dilution method — direct quantification of colonies of LAB in the biofilm [19; 20]. For direct quantitative colony counting of LAB in the pellicle, the method ofserial dilution was used, with, as substrate, two materials on whose surfaces LAB form a biofilm in vitro: coverslips and polystyrene plates. For this purpose, daily culture was grown on MRS medium. Then, the polystyrene and coverslip plate were placed on a sterile "edge" in weighing bottles (2x2 cm). Weighing bottles containing a cover glass and polystyrene plate were filled with 90% ethanol for 45 min, then the alcohol was decanted and the weighing bottle was rinsed with distilled water. It was dried at 37 °C for 30-40 min in sterile thermostat. In total, 4 ml of sterile MRS medium and 0.1 ml of daily culture oflactobacilli were added to the sterilized, dried weighing bottles with a cover glass and polystyrene plate. They were incubated at 37 °C for 24 h. Then, the culture fluid was gently poured out by tilting the weighing bottle. The LAB biofilm-coated cover glass and polystyrene plate were removed with sterile forceps and placed separately in sterile weighing bottles, to which 2 ml of saline were added. Repeatedly pipetting the saline completely washed away the biofilms of lactobacilli from the surface of the cover glass and polystyrene plate. We prepared 10-fold serial dilutions from each biofilm sample and plated them on dense medium MRS-agar. Cells were incubated at 37 °C for 24-36 h, followed by counting the colonies at various dilutions. The average was determined in CFU/ml. The results allow the determination of the ratio of viable colonies on the glass and polystyrene surfaces. The number ofviable of LAB indicates the biofilm forming activity of different strains.
Results
Antagonistic activity of LAB
The most antagonistic activity was showed against S. aureus and E. coli, the percentage of active strains LAB was 100% and 92% respectively. Cultures of RCM strains L. casei T B-RKM 0004, L. casei 3 B-RKM 0008, L. brevis 3-9 B-RKM 0010, L. fermentum 90T C 4-pl B-RKM 0014, L. plantarum 8RA-3 pl+ B-RKM 0015, L. plantarum pl-38 2/T B-RKM 0017, L. fermentum ATCC 9338 B-RKM 0018, L. casei L B-RKM 0027, L. delbrueckii subsp. lactis Cg-1 B-RKM 0044, and L. fermentum 136 B-RKM 0103 had high antagonistic activity toward all investigated test strains; the diameter of the zones of inhibition was range 10-13 mm. Antagonistic activity of LAB in relation to the test cultures is shown in Figure 1.
Figure 1. Antagonistic activity of LAB. The numbers in the wells 1, 2, 3, 4, 5 - replicate of test strain
The most antagonistic activity of the studied strains showed against enteric bacteria (E. coli) and yeast (C. albicans), where the zones of inhibition ranged from 5.5 to 13.0 mm: Lactobacillus casei 3 B-RKM 0008, L. casei L B-RKM 0027, L. delbrueckii CG-1 B-RKM 0044, L. fermentum B-RKM 0203, L. fermentum 90T C4-pl B-RKM 0014, L. brevis L9 B-RKM 0348, L. fermentum ATCC 9338 B-RKM 0018, L. brevis 3-9 B-RKM 0010, L. casei BI005 B-RKM 0208, L. casei GV B-RKM 0004, L. plantarum pl-38 B-RKM 0017.
All investigated collection strains and working isolates showed an average or high degree of antagonism toward at least some investigated test cultures. However, strains L. plantarum 2 B-RKM 0152, L. fermentum 96 B-RKM 0155, L. fermentum B-RKM 0203, L. casei BI 005 B-RKM 0208, L. brevis L5 B-RKM 0347, and L. brevis L9 B-RKM 0348 did not show an inhibitory effect toward S. marcescens. L. sakei 24a B-RKM 0559, L. sakei 7a B-RKM 0636, Lc. garvieae 10a B-RKM 0639, L. sakei 2a B-RKM 0640, P. pentosaceus 1a, Ln. garlicium 3a, Lc. lactis 14a, and Lc. lactis 17a showed higher antagonistic activity toward the bacterial test strains, than toward the yeast C. albicans.
The index ofviability of strains L. fermentum 90T C4-pl B-RKM 0014, L. sakei 2a B-RKM 0640, and L. sakei 7a B-RKM 0636, is stated only in the eighth breeding; Strain L. casei G B-RKM 0004 differs in the highest rates of viable colonies, 29.7x10 10 CFU/ml on coverslips.
While evaluating biofilm formation on a cover glass surface, it was found that L. fermentum 90T C4-pl B-RKM 0014, L. brevis L5 B-RKM 0347, L. delbrueckii Cg-1 B-RKM 0044, L. casei L B-RKM 0027, L. fermentum B-RKM 0203, Ln. garlicium 3a, L. sakei 7a B-RKM 0636, Lc. lactis 14a, and L. sakei 24a B-RKM 0559 showed little biofilm-formation (maximum rate of viability < 10 6 CFU/ml). Overall, the highest indicator of viability was L. plantarum 8RA-3 pl+ B-RKM 0015; it amounted 38x10 10 CFU/ml, on a polystyrene plate.
In terms of the different surfaces that were tested, it was found that L. plantarum 8RA-3 pl + B-RKM 0015 most actively formed a biofilm on the surface of polystyrene, and strain L. casei T B-RKM 0004 on the surface of a cover glass.
Determination of the biofilm-forming activity of LAB
Lactobacilli may actively form biofilms on the mucosal surface. Biofilm-formation, as well as the adhesive activity of lacto-bacilli, is regarded as a factor that enhances the colonization resistance of the resident intestinal microflora, and increases their probiotic properties and adaptation. Table 1 shows the results of in vitro biofilm formation in 10 10 dilution for the 25 LAB strains we tested. The index of maximum viability strains L. delbrueckii C-1 B-RKM 0044, L. casei L B-RKM 0027, L. fermentum B-RKM 0203, Lc. garlicium 3a, and Lc. lactis 14a, was <10 6 CFU/ml; thus the biofilm formatting activity was low and the data are not included in Table 1.
Biofilm formation on the surface of a cover glass was found for strains L. casei G B-RKM 0004, L. brevis 3-9 B-RKM 0010, L. plantarum 8RA-3 pl+ B-RKM 0015, L. plantarum pl-38 2/T B-RKM 0017, L. fermentum ATCC 9338 B-RKM 0018, L. fermentum 96 B-RKM 0155, L. fermentum 136 B-RKM 0103, L. plantarum 2B B-RKM 0152, and P. pentosaceus 8a, maximum indicators of viability were observed in the dilutions 10 8-10 10 CFU/ml.
Discussion
There is growth in the production of probiotics all over the world, as they are popular among people who support their health by using natural remedies; probiotic preparations are used in the food industry and medicine as pharmaceuticals or dietary supplements. Lactobacilli have been extensively investigated for probiotic activity, especially antagonism toward pathogenic microorganisms.
Scientists from Mongolia [21] screened 543 isolates of LAB derived from national dairy products in Mongolia. Investigated strains were tested for tolerance to gastric juice and bile acids, gassing and adhesion to Caco-2 cells. As a result, 10 prospective homofermenta-tive probiotic strains of LAB were selected and identified as L. plantarum and L. paracasei spp. Heterofermentative L. fermentum were also obtained, which can be used as starters for producing dairy products.
Jalilsood et al. [22] identified a new isolate of L. plantarum PA21, that could form a strong biofilm in pure culture and in combination with pathogenic and food-spoiling bacteria, such as Salmonella enterica, B. cereus, Pseudomonas fluorescens and Aeromonas hydrophila. Exposure to Lb. plantarum PA21 significantly reduced the number
Table 1. - Viability Indicator of lactic acid bacteria on polystyrene and coverslips
Strains of LAB Number of strain B-RKM Viability Indicator, CFU/ml, 10 10
polystyrene coverslips
L. casei Г 0004 4.0±1.0 29.7±1.03
L. fermentum 96 0155 12.0±4.0 13.0±0.88
L. plantarum 2 0152 4.5±1.50 12.0±2.0
L. fermentum ATCC 9338 0018 11.0±2.0 10.5±4.50
L. plantarum pl-38 2/T 0017 6.7±0.67 14.5±2.50
L. plantarum 8RA-3 pl 0015 38.0±1.0 2.0±1.0
L. fermentum 136 0103 29.5±0.50 10.0±5.0
L. brevis 3-9 0010 13.5±1.03 8.0±2.16
L. casei 3 0008 7.5±2.50 3.0±0.25
L. brevis L5 0347 1.0±0.50 -
L. casei BI 005 0208 1.0±0.50 3.0±0.25
L. brevis L9 0348 2.0±0.17 1.0±0.50
P. pentosaceus 1 а - 9.0±2.42 2.5±0.96
L. sakei 2 а 0640 - 1.7±0.67
P. pentosaceus 8a 0635 19.7±4.66 11.2±4.99
L. garvieae 10 а 0639 5.2±0.63 3.5±0.50
L. lactis 17a - 8.2±1.70 5.5±1.50
L. sakei 24 а 0559 5.0±1.0 -
of P. fluorescens, A. hydrophila and B. cereus cells in the biofilm over 2-, 4-and 6-days. However, despite a reduction in the number of S. enterica cells, this pathogen showed greater resistance to the Lactobacillus plantarum PA21, either in the planktonic or biofilm phase. Lb. plantarum PA21 was also found to be able to constitutively express GFP when transformed with the expression vector pMG36e, which harbors the gfp gene as a reporter, demonstrating that the newly isolated strain could be used as host for genetic engineering.
Researchers worldwide spend search of strains of LAB that have antagonistic and biofilm forming activity for various applications in the food and pharmaceutical industries.
The advantage and novelty of the present research lies in the fact that it screened active strains ofLAB not only for antagonism to opportunistic pathogens, but for biofilm-forming ability. Bacteriocin produc-
tion, along with the production of for example, lactic acid, hydrogen peroxide, and lysozyme, relates to antagonism [23, 24, 25] biofilm formation determines the ability of microorganisms to actively colonize the mucosal surfaces of an organism. The biological characteristics of biofilm formation in S. aureus have been investigated as a factor that enhances their pathogenic potential, and in lactobacilli as a factor that enhances their ability to colonize the vaginal biotope [26, 27].
The present research revealed that the best among the tested species in biofilm-formation were L. plantarum 8^.-3 pl + B-RKM 0015 and L. casei T B-RKM 0004, which actively colonized the surfaces of polystyrene plates and cover glass, respectively. During study of the antagonistic activity of all LAB strains, it was found that L. casei 3 B-RKM 0008 had quite a strong antimicrobial effect on all test cultures.
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