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17
CHARACTERIZATION OF THE ADHESIVE PROPERTIES OF BACILLUS INTESTINALIS GM2
A.A. Nikolaeva*, G.F. Lutfullina, A.M. Mardanova
Kazan Federal University, 18 Kremliovskaya St., Kazan, 420008, Russia.
* Corresponding author: azazel1212@rambler.ru
Abstract. In this paper, we present the data on the properties of the probiotic strain Bacillus intestinalis GM2 related to adhesion, auto-, and coaggregation. GM2 exhibits a strong autoaggregation phenotype. The autoaggregation ability of GM2 did not change after trypsin treatment but was reduced under the influence of extracellular culture metabolites. Coaggregation of probiotic strains with pathogens is one of the mechanisms of displacement of pathogens from the intestinal microbiota. It was shown that the GM2 strain exhibits the ability to coaggregate with tested strains of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Bacillus cereus. The studied properties of bacilli can be useful for the creation of new probiotics for poultry farming.
Keywords: probiotics, Bacillus subtilis GM2, adhesion, autoaggregation, coaggregation, biosurfactants.
List of Abbreviations
PBS - phosphate-buffered saline
LB - lysogeny broth
SMN - soybean medium nutrition
Introduction
To improve the performance of farm animals, nutritional measures are developed, primarily aimed at changing the functions of the gastrointestinal tract. Probiotics and prebiotics are potential alternatives to antibiotics in livestock production. Probiotic strains limit direct contact of pathogenic bacteria with the gut epithelium by competitively excluding nutrients and surfaces, as well as by creating a hostile environment and producing bioactive compounds acting selectively on pathogens (Binns et al, 2013; Celi et al, 2019; Peralta-Sánchez et al, 2019).
To exhibit beneficial effects, it is vital that probiotic bacteria achieve a specific cellular concentration through aggregation (Collado et al, 2008). Autoaggregation, or autoagglutination, is one of the means, by which cells adapt to environmental conditions. By binding bacteria to each other, it increases the relative adaptability of aggregates compared to single cells in the space and resource-constrained environment that is the gut environment (Kragh et al, 2016; Isenring et al, 2021; Nwoko & Okeke, 2021). In addition, autoaggregation facilitates
biofilm formation, which also enhances adaptability to the environment through increased cell density and cell-to-cell interaction (Giaou-ris et al., 2015; Laganenka et al., 2016; Kragh et al, 2016). Nonetheless, it has been reported that coaggregation does not always lead to biofilm formation (Hiramatsu et al., 2016).
The genus Bacillus includes rod-shaped, en-dospore-forming, saprophytic soil aerobes. Several strains of the Bacillus genus have been isolated from the feces of broiler chickens, which suggests their presence in the intestines of birds (Barbosa et al., 2005). The ability of these microorganisms to proliferate and sporulate in the gut suggests a high potential for transit between the soil (natural reservoir) and the gut. It has been confirmed that Bacillus spores are able to germinate in the small intestine and interact with enterocytes, immunocompetent cells, and/or the intestinal microbiota of the host (Barbosa et al, 2005; Menconi et al, 2013).
Biosurfactants have both hydrophilic and hydrophobic moieties, causing aggregation at interfaces between liquids with different polarities, such as hydrocarbons and water. Thus, they reduce surface and interfacial tension as well as form emulsions (Sarwar et al, 2018). Compared to chemical or synthetic surfactants, biosurfactants offer several advantages, including high biodegradability, low toxicity, and effectiveness at extreme temperatures or over a
wide pH range. Lipopeptides rank first among the numerous classes of biosurfactants. The interest in lipopeptides is mainly due to their high surface activity and antimicrobial potential (Vaz et al., 2012). Bacillus strains are known to produce a wide range of lipopeptides: fengicins, iturins, mycosubtilins, bacillomicins, and sur-factins with great potential for biotechnology and biopharmaceutical industries (Perez et al, 2017). Biosurfactants change the hydrophobi-city of the bacterial surface and therefore are able to regulate the adhesion of microorganisms to solid surfaces. The involvement of bacterial biosurfactants in microbial adhesion and desorption has been extensively described by Rodrigues et al. (Rodrigues et al, 2011). The pre-adsorption of biosurfactants on solid surfaces can be an effective strategy to reduce microbial adhesion and prevent colonization by pathogens (Gudina et al, 2010).
We have previously shown that the Bacillus intestinalis GM2 strain exhibits high antagonistic activity against pathogenic and opportunistic enterobacteria, resistance to bile and a wide range of pH environments, as well as the ability to synthesize proteolytic and phytate hydrolyz-ing enzymes (Hadieva et al, 2018). In addition, in vivo studies have shown a positive effect of a feed additive based on B. intestinalis GM2 spores in the diet of Cobb-500 broiler chickens on growth performance and nutrient digestibility (Hadieva et al, 2019).
This study aimed to evaluate the adhesive properties of the B. intestinalis GM2 strain and its ability to produce biosurfactants and to co-aggregate with strains of opportunistic bacteria.
Materials and Methods
Bacterial strains and growth conditions
Strains of B. intestinalis GM2 and B. subtilis GM5 with high antagonistic activity were isolated from the potato rhizosphere (Mardanova et al, 2017). B. subtilis strain VKPM B-10641, which was isolated from the commercial probi-otic Vetom 1.1, was used for comparative assays. For comparative analysis of coaggrega-tion, the following strains were used: Esche-richia coli isolated from the urethra and provided by the clinical and diagnostic laboratory
BIOMED of Kazan, as well as strains of Enter-ococcus faecalis, Staphylococcus aureus, and Bacillus cereus obtained from the culture collection of the Department of Microbiology of KFU.
Bacteria were cultured in LB (Lysogeny broth) medium (g/l): tryptone - 10.0, yeast extract - 5.0, NaCl - 5.0; LA medium (g/l): tryptone - 10.0, yeast extract - 5.0, NaCl - 5.0, agar - 20.0. To study the synthesis of biosurfactants by B. intestinalis GM2 strain, soybean medium nutrition (SMN) (g/l): mannitol - 26.2, soybean meal - 21.9, NaNOs - 3.1, MnSO4x4№O - 0.2 (pH 7.5) was used. Bacteria were cultured at 37 °C. When necessary, cultures were maintained in a shaker thermostat at 37 °C and aerated at 200 rpm.
Autoaggregation and coaggregation assays
The study was performed as described in (Kos et al., 2003) with certain modifications. Night culture cells were harvested by centrifu-gation, washed twice, and resuspended in phosphate-buffered saline (PBS) or in culture supernatant fluid to obtain OD600 = 0.3 ± 0.05 (107108 CFU/ml). The cell suspension (3 ml) was mixed for 10 s and then incubated at room temperature for 24 h without aeration. The optical density (OD600) of the top of the suspension was measured after 2, 4, 6, and 24 hours of incubation on a spectrophotometer (Bio-Rad xMark, U.S.A.) The percentage of autoaggregation was expressed as:
A (%) = 1 - (At / A0) x 100,
where At is the optical density at time t = 2, 4, 6, and 24 h, and A0 is the optical density at 0 h.
The method of preparing the bacterial suspension for the coaggregation assay was the same as in the autoaggregation assay. Equal volumes (1.5 ml) of each bacterial cell suspension for coaggregation were mixed in pairs by shaking for 10 s. The control variants each contained 3 ml of a suspension of axenic bacterial cultures. The absorbance (A) at 600 nm was measured immediately after mixing, at 4 and 24 h of incubation at room temperature. The percentage of coaggregation was calculated as follows:
Ao (%) = [(Apat + Aprobio)/2 -- (Amix)/(Apat + Aprobio)/2]x 100,
where Apat and Aprobio represent A 600 of the separate bacterial suspensions in control tubes at 0 h, and Amix represents the absorbance of the mixed bacterial suspension (Collado et al, 2008).
Trypsin treatment of bacterial cells For trypsin treatment, overnight culture cells were washed in PBS, then 100 pl of 10x trypsin was added to 900 pl (OD600 = 1) of the suspension, cultured at 37 °C for 1 hour, then treated as described above.
Adhesion ability to polystyrene To study the adhesive properties, overnight culture cells were diluted with fresh sterile medium to OD600 = 0.5, then 1.0 ml of diluted cultures were introduced into polystyrene Petri dishes (4 cm diameter), which were incubated for 4 h at 30 °C. Next, non-adherent cells were drained and washed with sterile saline. Adherent bacteria were stained with gentian violet solution for 2 min and then the nonadherent dye was washed off. E. coli and S. aureus strains were used to study coadhesion and coaggregation of B. intestinalis GM2 in mixed culture. Equal volumes (0.5 ml) of each cell suspension were mixed by suspension and introduced into polystyrene Petri dishes. After incubation and staining, they were microscopically examined using the MICROS AUSTRIA MC 300 (Austria) equipment at a magnification of 100x.
Measurement of surface tension The dynamics of biosurfactant synthesis by B. intestinalis GM2 strain were tested by reducing the surface tension of the culture medium. The change in surface tension was determined by the Dubois Ring method, as described earlier (Rodriguez et al., 2006). The surface tension of the medium was measured on a BT-500 torsion scale (Russia) equipped with a platinum ring with a diameter of 1.9 cm. The surface tension values represent the average value.
Statistical Analysis
All experiments were performed in triplicate. Data were evaluated by ANOVA with repeated measures with a significance level of P < 0.05. Results are presented as mean ± standard deviation.
Results
Comparative characterization of the sedimentation rate of the three Bacillus strains showed that strain GM2 exhibited the strongest autoaggregation ability within 24 hours as compared with the probiotic strains B. subtilis GM5 and B. subtilis VKPM B-10641 (Fig. 1). The GM2 strain also showed a visually more transparent supernatant even at 2 h of incubation relative to the other strains.
Fig. 1. Comparison of the autoaggregation ability of different probiotic Bacillus strains: Bacillus intestinalis GM2 (o), Bacillus subtilis GM5 (■) Bacillus subtilis VKPM B-1064 (▲)
The observed autoaggregation was not lost after washing and suspending the cells GM2 in PBS. At the same time, extracellular metabolites reduced the ability of the strain to form aggregates (Fig. 2), since the autoaggregation activity was significantly reduced (p < 0.05) when the cells were resuspended in the culture fluid in which they were initially growing. There was no significant difference in the ability to autoaggregate after treatment of the cells with trypsin.
Autoaggregation and coaggregation are important for biofilm formation by probiotic strains to protect the host from colonization by pathogens (Botes et al, 2008).
It was previously shown that in contrast to 37 °C, 20 °C was a more suitable temperature
10CH
OfS- -1-1-1-1
0 2 4 6 24
Time (h)
Fig. 2. Comparative analysis of the autoaggregation ability of Bacillus intestinalis cells resuspended in PBS (pH 7.2) (o) after trypsin treatment (■) or resuspended in their own culture supernatant fluid (▲)
Table 1
Coaggregation capacity of Bacillus intestinalis GM2 with potential pathogenic bacteria
at room temperature in PBS (pH 7.2)
Coaggregation with B. intestinalis GM2 (%)
4 hour 24 hour
Mean SD Mean SD
E. coli 35,46 2,32 42,96 3,15
S. aureus 20,81 3,62 48,76 4,44
B. cereus 26,05 0,76 73,3 1,49
E. faecalis 12,33 1,57 49,2 3,89
when analyzing the ability of bacteria to coag-gregate (Collado et al, 2008). Therefore, the coaggregation ability of the GM2 strain was tested at room temperature (Table 1). The GM2 strain showed the ability to coaggregate with the tested strains of potential pathogens, and the percentage of coaggregation depended on the incubation time. The highest percentage of bacterial coaggregation at 4 hours of incubation was with cells with E. coli (35.5%) and the lowest with E. faecalis (12.3%). At 24 hours the percentage of co-aggregation with B. cereus reached 73%, while with E. coli it did not exceed 43%. Thus, the GM2 strain showed the ability to coaggregate with all tested pathogens, but the efficiency of coaggregation depended on the test culture strain and the time of co-incubation.
The adhesive ability of the GM2 strain on the polystyrene surface was studied (Fig. 3). GM2 cells in the culture medium (LB) were attached in moderate numbers, with adhesion to the surface occurring in small groups with close contact between individual cells (Fig. 3a). A large percentage of the surface was covered with culture at 4 hours of incubation, and cell morphology was well traceable (Fig. 3b). Tryp-sin treatment did not reduce the adhesive ability of GM2. On the contrary, cell adhesion was observed in large aggregates (Fig. 3 c, d) in contrast to GM2 cells in nutrient broth, where adhesion to the surface was observed more often in small groups of cells. The inhibition of adhesive ability for cells suspended in PBS was observed in the early hours of cultivation (Fig. 3e). GM2 cells in PBS formed large aggregates at
Fig. 3. Adhesion of B. intestinalis GM2 to the surface of polystyrene petri dishes after 2h (a, c, e) and 4h (b, d, f) incubation. B. intestinalis GM2 cells in LB medium (a, b), after trypsin treatment (c, d) or resuspended in PBS (pH 7.2) (e, f). Staining with gentian violet, magnification x1600. The bar corresponds to 5 pm
Fig. 4. Coadhesion of B. intestinalis GM2 to the surface of polystyrene petri dishes after 2h (a, c, e) and 4h (b, d, f) incubation. B. intestinalis GM2 cells in LB medium (a, b), GM2 and Staphylococcus aureus (c, d) or GM2 and Escherichia coli (e, f). Staining with gentian violet, magnification x1600. The bar corresponds to 5 pm
Fig. 5. Growth dynamics and changes in surface tension of culture fluid of B. intestinalis GM2 on SMN medium (■)
4 h, in which they adhered to the polystyrene surface (Fig. 3f). The presence of a large number of multilayer aggregates was noted. At the same time, GM2 cells were smaller in size, which may be due to nutrient deficiencies.
In the case of mixed GM2 and S. aureus cultures, individual clusters of cells were found on the polystyrene surface during microscopy at 2 hours of incubation, but at 4 hours, large multilayer bacterial aggregates were noticed (Fig. 4c, d). Moreover, the formation of these aggregates enhanced cell adhesion. Also during coincuba-tion of GM2 and E. coli cultures, the formation and adhesion of coaggregates on the polystyrene surface were observed as early as after 2 h, which significantly increased in number after 4 h (Fig. 4e, d). Thus, (Fig. 4a, b), the coincu-bation of Bacillus cells with both test bacteria significantly increased the ability of bacteria to aggregate, coaggregate, and consequently, adhere to a solid surface in comparison with GM2 monoculture.
We studied the dynamics of bacterial growth and accumulation of biosurfactants in SMN medium exploring extracellular metabolites of B. intestinalis GM2 exhibiting anti-adhesion activity. When GM2 was cultured at 37°C on
SMN medium, a stationary growth phase was observed after 24 hours of growth (Fig. 5). The late shift to the stationary phase was due to the long period of the log phase during which the bacteria adapted to the conditions of the cultivation. The rationale for the inclusion of soybean meal in the GM2 strain cultivation medium was based on the use of this substrate in the industrial production of surfactants as a nitrogen-containing raw material. Soybean meal contains a balanced composition of proteins and carbohydrates that are necessary for the growth and synthesis of secondary metabolites (Zhang et al., 2016). Accumulation of biosur-factants by GM2 strain in the culture medium was determined by a decrease in surface tension. At the initial point, the surface tension index was at 88-90 mN/m. The surface tension was reduced after 12 h of cultivation (Fig. 5) and remained at 36 mN/m at 30 h, indicating a high level of biosurfactant synthesis by the GM2 strain. It was shown that the synthesis of biosurfactants in the cultivation medium did not negatively affect the growth of the B. intesti-nalis GM2 strain, since the proliferation and accumulation of bacterial biomass in the culture continued for 48 h. Thus, a decrease in the sur-
face tension of the cultivation medium indicates efficient synthesis of biosurfactants by B. intes-tinalis GM2 culture.
Discussion
The ability to adhere to epithelial cells and mucosal surfaces is considered an important property of many bacterial strains used as pro-biotics. In most cases, the ability to aggregate is related to the adhesive properties of the cells (Kos et al, 2003). The relationship between autoaggregation and adhesion ability has been reported for some bifidobacterial species (Del Re et al, 2000). It has been shown on lactobacilli how surface-mediated properties of bacteria, such as aggregation, can have a role in the adhesion and colonization of the gastrointestinal tract (Cesena et al, 2001). On the other hand, other studies have found no correlation between aggregation and adhesion to Caco-2 cells (Botes et al., 2008). These discrepancies can be explained by differences in methods. The GM2 strain exhibited a strong auto-aggregation capacity that was not lost after washing and suspending the cells in PBS and reached more than 80% at 24 h, which is consistent with studies by other authors. For instance, it has been reported that the percentages of 93.42 ± 0.86%; 86.03 ± 2.46% and 91.32 ± 0.74% were recorded for B. clausii ATCC 700160, B. subtilis P223, and B. subtilis MKHJ 1-1 after 24 hours of incubation, respectively (Jeon et al., 2017; Lim et al., 2021). In another study, the autoaggregation activity of several probiotic strains of Bifidobac-terium and LactoBacillus was examined; the percentage of autoaggregation at 24 hours ranged from 31.2 ± 2.2% (B. breve 99) to 76.4 ± 8.3% (L. plantarum Lp-115) (Collado et al, 2008).
Autoaggregation and coaggregation are known to have an important role in biofilm formation to protect the host from colonization by pathogens (Botes et al., 2008). The relationship between the coaggregation abilities of bacteria and autoaggregation properties has been shown previously (Collado et al, 2008). It has been suggested that probiotic bacteria, by coaggre-gating with pathogens, produce antimicrobial compounds, or remove pathogens from the gut
environment as part of coaggregates, which depicts an important mechanism for protecting the gut from infection (Spencer et al., 1994; Botes et al., 2008). The GM2 strain has been shown to be capable of coaggregating with potential pathogens, which may be useful when used as probiotic cultures.
Bacterial surface adhesion is influenced by various factors including microbial type, hydro-phobicity, surface electrical charges, environmental conditions, and the ability of microorganisms to produce extracellular polymers that help cells attach to surfaces (Vijayakumar & Saravanan, 2015). Bacterial biosurfactants can be used to change the hydrophobicity of the surface, which affects bacterial adhesion. Chakrabarti et al., showed that a biosurfactant isolated from the culture fluid of Streptococcus thermophilus inhibits the colonization of other thermophilic Streptococcus strains. Biosurfac-tant produced by the bacterium Pseudomonas fluorescens inhibited the attachment of Listeria monocytogenes to the steel surface (Chakrabarti, 2012). A biosurfactant isolated from the supernatant of B. subtilis EG1 exhibits anti-adhesive activity against S. aureus and E. coli. The highest anti-adhesion percentage was obtained for S. aureus (28.6%) at a biosurfactant concentration of 3000 mg/L. Similar studies of the antiadhesive properties of surfactin on polystyrene surfaces were carried out (Zeraik & Nitschke, 2010). The authors showed that S. au-reus attachment to polystyrene could be reduced by 42.2% by surface conditioning with surfactin. E. coli CFT073 and S. aureus ATCC 29213 biofilm formation was also shown to be reduced by 97% and 90%, respectively, by the presence of surfactin in the medium (Rivardo et al, 2009). Thus, the decrease in autoaggregation activity of the GM2 strain, as shown in Fig. 2, may be due to the synthesis of biosurfactants in the culture medium.
The GM2 strain showed a high ability to adhere to polystyrene surfaces. Adhesive and aggregation properties have been shown to depend on environmental conditions. Apparently, microbial aggregation ability is enhanced under stress conditions, as the largest bacterial aggregates were noted during nutrient deficiency (in
phosphate-buffered saline), which enhanced cell adaptation due to higher spatial positioning (Kragh et al, 2016).
Adhesion assays of axenic GM2 culture and mixed cultures of GM2 with other gram-positive and gram-negative bacteria showed that co-incubation of different species affects the ability of bacteria to form aggregates and exhibit adhesive properties (Table 1 and Fig. 4). Thus, the presence of E. coli and S. aureus has a positive effect on GM2 adhesion by promoting the formation of multilayer aggregates (Fig. 4). It can be noted that a comparative assay of bacterial coaggregation at 4 h of incubation showed a greater ability of GM2 cells to interact most strongly with E. coli. The effect of one bacterial species on the adhesion of another bacterial species has been shown previously, with both positive and negative effects on adhesion being observed. The ability of bacterial cells to adhere to the surface and each other can be affected by the secretion of poly-
mers, proteins, surfactants, and metabolites by microorganisms present in suspension or on the surface (Cutter et al., 2003). Also, despite similar physicochemical mechanisms of coad-hesion and coaggregation, coadhesion of coag-gregating bacterial pairs does not always occur (Rolf et al, 1999).
In conclusion, the B. intestinalis GM2 strain showed high adhesive properties, which could potentially be useful in the gastrointestinal environment. Additional studies of the bacteria's ability to aggregate under conditions simulating the avian gastrointestinal tract conditions are needed.
Acknowledgments
This study was supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030). This work was funded by the grant RFBR-20-34-90130. The authors thank Y.A. Akosah for helping with the language editing of the manuscript.
References
BARBOSA T.M., SERRA C.R., LA RAGIONE R.M., WOODWARD M.J. & HENRIQUES A.O. (2005): Screening for bacillus isolates in the broiler gastrointestinal tract. Applied and environmental microbiology 71(2), 968-978.
BINNS N. (2013): Probiotics, prebiotics and the gut microbiota. ILSIEurope Concise Monograph. Belgium, 1-32.
BOS R., VAN DER MEI H.C. & BUSSCHER H.J. (1999): Physico-chemistry of initial microbial adhesive interactions—its mechanisms and methods for study. FEMS microbiology reviews 23 (2), 179-230.
BOTES M., LOOS B., VAN REENEN C.A. & DICKS L.M. (2008): Adhesion of the probiotic strains En-terococcus mundtii ST4SA and Lactobacillusplantarum 423 to Caco-2 cells under conditions simulating the intestinal tract, and in the presence of antibiotics and anti-inflammatory medicaments. Archives of microbiology 190(5), 573-584.
CELI P., VERLHAC V., PÉREZ CALVO E., SCHMEISSER J. & KLUENTER A. (2019): Biomarkers of gastrointestinal functionality in animal nutrition and health. Animal Feed Science and Technology 250, 9-31.
CHAKRABARTI, S. (2012): Bacterial biosurfactant: Characterization, antimicrobial and metal remediation properties. Ph.D. Thesis, National Institute of Technology.
COLLADO M.C., MERILUOTO J. & SALMINEN S. (2008): Adhesion and aggregation properties of probiotic and pathogen strains. European Food Research and Technology 226, 1065-1073.
CUTTER L.A., VAN SCHIE P.M. & FLETCHER M. (2003): Adhesion of anaerobic microorganisms to solid surfaces and the effect of sequential attachment on adhesion characteristics. Biofouling 19, 18-9.
GIAOURIS E., HEIR E., DESVAUX M., HÉBRAUD M., M0RETR0 T., LANGSRUD S., DOULGERAKI A.I., NYCHAS G.E., KACÁNIOVÁ M., CZACZYK K., OLMEZ H. & SIMOES M. (2015): Intra- and inter-species interactions within biofilms of important foodborne bacterial pathogens. Frontiers in Microbiology 6.
GUDIÑA E.J., ROCHA V., TEIXEIRA J A. & RODRIGUES L.R. (2010): Antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillusparacasei ssp. paracasei A20. Letters in applied microbiology 50(4), 419-424.
HADIEVA G.F., LUTFULLIN M.T., MOCHALOVA N.K., SHARIPOVA M.R., MARDANOVA A.M. & LENINA O.A. (2018): New Bacillus subtilis strains as promising probiotics. Microbiology 87 (4), 463-471.
HADIEVA G.F., LUTFULLIN M.T., NIKOLAEVA A.A., MOCHALOVA N.K., SMOLENCEV S.Y., MARDANOVA A.M. & SHARIPOVA M R. (2019): The effect of Bacillus subtilis GM2 and GM5 probiotics on the growth and fodder digestibility of broiler chickens. Uchenye Zapiski Kazanskogo Univer-siteta. Seriya Estestvennye Nauki 161 (3), 472-489.
HIRAMATSU Y., SAITO M., OTSUKA N., SUZUKI E., WATANABE M., SHIBAYAMA K., & KAMA-CHI K. (2016): BipA Is Associated with Preventing Autoagglutination and Promoting Biofilm Formation in Bordetella holmesii. PLoS ONE 11.
ISENRING J., GEIRNAERT A., LACROIX C., & STEVENS M.J. (2021): Bistable auto-aggregation phe-notype in Lactiplantibacillus plantarum emerges after cultivation in in vitro colonic microbiota. BMC Microbiology 21.
KOS B., SUSKOVIC J., VUKOVIC S., SIMPRAGA M., FRECE J. & MATOSIC S. (2003): Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. Journal of Applied Microbiology 94.
KRAGH K.N., HUTCHISON J.B., MELAUGH G., RODESNEY C.A., ROBERTS A.E., IRIE Y., JENSEN P.0., DIGGLE S.P., ALLEN R.J., GORDON V.D. & BJARNSHOLT T. (2016): Role of Multicellular Aggregates in Biofilm Formation. mBio 7.
LAGANENKA L., COLIN R. & SOURJIK V. (2016): Chemotaxis towards autoinducer 2 mediates autoaggregation in Escherichia coli. Nature Communications 7.
MARDANOVA A., HADIEVA G., LUTFULLIN M., KHILYAS I.V., MINNULLINA L., GILYAZEVA A G., BOGOMOLNAYA L.M. & SHARIPOVA M.R. (2017): Bacillus subtilis Strains with Antifungal Activity against the Phytopathogenic Fungi. Agricultural sciences 08, 1-20.
MENCONI A., MORGAN M.J., PUMFORD N.R., HARGIS B. & TELLEZ G. (2013): Physiological Properties and Salmonella Growth Inhibition of Probiotic Bacillus Strains Isolated from Environmental and Poultry Sources. International Journal of Bacteriology 2013.
NWOKO E.Q. & OKEKE I.N. (2021): Bacteria autoaggregation: how and why bacteria stick together. Biochemical Society Transactions 49, 1147-1157.
PERALTA-SÁNCHEZ J.M., MARTÍN-PLATERO A.M., ARIZA-ROMERO J.J., RABELO-RUIZ M., ZURITA-GONZÁLEZ M.J., BAÑOS A., RODRÍGUEZ-RUANO SM., MAQUEDA M., VALDIVIA E. & MARTÍNEZ-BUENO M. (2019): Egg Production in Poultry Farming Is Improved by Probiotic Bacteria. Frontiers in Microbiology 10.
PEREZ K. J., SANTOS VIANA J. DOS, LOPES FC., PEREIRA J.Q., SANTOS D.M. DOS, OLIVEIRA J.S., VELHO R.V., CRISPIM S M., NICOLI J. R., BRANDELLI A. & NARDI, R.M.D. (2017): Bacillus spp. Isolated from Puba as a Source of Biosurfactants and Antimicrobial Lipopeptides. Front Microbiol. 8.
RIVARDO F., TURNER R.J., ALLEGRONE G., CERI H. & MARTINOTTI M.G. (2009): Anti-adhesion activity of two biosurfactants produced by Bacillus spp. prevents biofilm formation of human bacterial pathogens. Appl. Microbiol. Biotechnol. 83 (541).
RODRIGUES L.R., IN: LINE D., GOLDMAN A. (EDS ). (2011): Bacterial Adhesion: Biology, Chemistry, and Physics, Book Series "Advances in Experimental Medicine and Biology". Springer.
RODRIGUES L.R., TEIXEIRA J.A., VAN DER MEI H.C. & OLIVEIRA R. (2006): Isolation and partial characterization of a biosurfactant produced by Streptococcus thermophilus A.Colloids Surf. B Biointerfaces 49 (78).
SARWAR A., BRADER G., CORRETTO E., ALETI G., ULLAH M. A., SESSITSCH A. & HAFEEZ FY. (2018): Qualitative Analysis of Biosurfactants from Bacillus Species Exhibiting Antifungal Activity. PLoS One 13 (6).
VAZ D A., GUDINA E.J., ALAMEDAA E.J., TEIXEIRA J A. & RODRIGUES L.R. (2012): Performance of a biosurfactant produced by a Bacillus subtilis strain isolated from crude oil samples as compared to commercial chemical surfactants. Colloids and Surfaces B: Biointerfaces 89, 167-174.
VIJAYAKUMAR S. & SARAVANAN V. (2015): Biosurfactants-Types, Sources and Applications. Research Journal of Microbiology 10(5), 181-192.
ZERAIK A.E. & NITSCHKE M. (2010): Biosurfactants as Agents to Reduce Adhesion of Pathogenic Bacteria to Polystyrene Surfaces: Effect of Temperature and Hydrophobicity. Curr. Microbiol. 61 (554). ZHANG J., XUE Q., GAO H., LAI H. & WANG P. (2016): Production of lipopeptide biosurfactants by Bacillus atrophaeus 5-2a and their potential use in microbial enhanced oil recovery. Microb Cell Fact. 15.
