Interaction of Probiotic Bacterial Lectins to Candida species

Biological Sciences/6.Microbiology

Dr. Lakhtin M., Dr. Lakhtin V., Dr. Bajrakova A., Dr. Aleshkin A.,

Dr. Prof. Аfanasiev S., and Dr. Prof. Aleshkin V.

G.N. Gabrichevsky Research Institute for Epidemiology and Microbiology, Russia

Interaction of Probiotic Bacterial Lectins to Candida species

Abstract: Responses of suspensions of clinically significant urogenital strains of Candida species to the presence of probiotic bacterial lectins PBL of human origin and antibiotics were compared. Anti-Candida species responses depended on type of PBL or antibiotics. Higher relative virulence of Candida species corresponded to higher anti-Candida species activity of PBL. PBL distinguished Candida species upon prolonged co-incubations. The role of PBL in biotopes is discussed.

Keywords: Probiotic lectins, Bifidobacteria, Lactobacilli, Antifungals, Candida, Urogenital tract.

Introduction: Candida infections are of increased interest of investigators. In spite of extended using antibiotics against Candida, alternative antimicrobials are required because of development of the resistance of pathogens to antibiotics.

Probiotic microbes support healthy status in human and can be used as the strategic direction in medical microbiology [1, 2]. Lectins (glycoconjugate recognizing ptoteins) are widely occurred in nature [3]. Probiotic bacterial lectins (PBL) possess activities of probiotics [4-6]. Lectins of lactobacilli and bifidobacteria (LL and LB) were identified, isolated and standardized [7-10]. A number of useful activities of PBL were described [6]. Among them, LL and LB reveal features of destructors of pathogen biofilms [10]. Activities against C. albicans clinical strains isolated from patient intestinal biotope were demonstrated.

The aim was further study of interaction between PBL and Candida species clinical strains.

Materials and methods

Strains and growth conditions: Industrial probiotic lactobacillus and bifidobacterial strains which serve as ingredients of probiotics were used as source of PBL (Table 1). Bacteria were grown for 18-24 h at 37^oC in standard liquid media (pH 7) [7].

Candida clinical strains were isolated from urogenital tract of patients observed in the Clinical Diagnostic Center at G.N. Gabrichevsky Research Institute for Epidemiology & Microbiology (www.gabrich.com). Characteristics of some clinical strains used are presented in Table 2. Candida species were identified using chromogenic agar media (HiMedia Lab. Pvt. Ltd., India). Stocks of identified Candida strains were grown on agar in standard conditions as discontinuous equally distributed on Sabouraud agar yeast suspension (10⁸ Cfu mL^-1) at 37^oC for 24-48 h.

Antimicrobials used: Standard panel of disc-antibiotics included Amphothericin B, Clotrimazol, Fluconazol, Itraconazol, Ketoconazol & Nystatin (HiMedia Lab. Pvt. Ltd., India).

Preparations of acidic lectins of lactobacilli and bifidobacteria (aLL and aLB) were isolated using cultural fluid supernatant fractionation followed by separation with isoelectric focusing in polyacrylamide gels as described earlier [7]. PBL interacted to mannans and mucin-like polymers. Preparations were free of oxidase-reductase system. Lectins were used in subhemagglutinating dilutions [10].

Assay of anti-Candida action of PBL: The growth of freshly isolated and identified Candida clinical strains on Sabouraud agar was performed in the presence of antimicrobial discs. Antifungal discs were distributed on agar in the center and peripheral regions in plates [10].

Anti-Candida strain action of discs was evaluated in the following manner. Firstly, antibiotic-like activity of PBL-disc was measured as for antibiotic-discs (the diameter of zone around disc which was free of growth. In order to confirm Candida species sensitivity to antifungals, the variability parameter was calculated: VP(%) = (D_max – D_min)/D_max x 100, where D= diameter of diffusion inhibition zone around the disc. Secondly, the distant (non-antibiotic-like) anti-Candida activity of PBL-discs was visually evaluated in the central and peripheral directions from discs [10].

Assay of interaction between Candida suspension and PBL: Interaction between PBL and suspensions of Candida strains was optically controlled in microplates. Suspensions of C. albicans,

C. glabrata, C. krusei or C. tropicalis strains were grown as noted above. Cells were harvested, washed in sterile 0.9% saline and diluted to 1.0 (MacFarland standard turbidity units). Equal volumes (100 µL) of prepared stocks were placed into wells of flat-bottomed 96-well microtiter plates (Biomedicals, Moscow). 100 µL of stock solution of PBL was added to the first wells containing Candida suspensions, and then series of wells containing PBL 2-fold-dilutions in 0.9% saline and equal volumes of Candida suspensions were prepared. Final volumes in microplate wells (100 µL) corresponded to 0.5 MacFarland standard turbidity units. Control wells contained additional volume of 0.9% saline instead of PBL solution. Suspensions were incubated in microplates for 24-72 h 37^oC in the presence of PBL (stock solution 1µg mL^-1 in dilutions 10-10,000 times) in 0.9% saline equally distributed in suspension volume, and cell optical density (OD) at 600 nm (OD₆₀₀) was periodically analyzed using microtiter plate reader Multiscan EX (ThermoLabsystems, Helsinki, Finland). Yeast suspension mixtures selected did not form optically detectable biofilms upon incubation (0.9% saline instead of cell suspensions in wells did not result in significant residual increase of OD₆₀₀). Similarity between responses of strains to the presence of PBL was evaluated by comparison of changing OD₆₀₀ in yeast suspensions during 3 days for all PBL dilutions tested.

Statistics: All studies were performed at least in triplicate times. A Student's t-test was used to compare calculated data, with a P < 0.05 representing a statistical significance.

Results

Candida species responses to PBL and antibiotics: The data on Interaction between PBL and Candida species are shown in Table 3. PBL revealed anti-Candida species activities. Candida species growth inhibition depended on type of PBL. Antibiotic and PBL activities against Candida species are represented in the Table 3. The effectiveness of antibiotics against Candida species was decreased in the order: [K > F] > С > I > A > N (for C. albicans), С > I > [K > F] > A > N (for C. krusei) or С >

[K > F] > I > A, N (for C. tropicalis). As a result, the relative Candida species sensitivity to antibiotics was varied by position of the block [Ketoconazol, Fluconazol]. In general, C. albicans cases reveal both maximal action levels of Ketoconazol, Fluconazol. On the other hand, cases of C. krusei were characterized by decreased responses to these antifungals. Results indicate relationship between Candida species sensitivities to PBL and antibiotics. It is seen that relatively more pathogenic species (C. albicans followed by non-albicans Candida species) corresponds to stronger anti-Candida species response to aLB, Fluconazol and Ketoconazol. Variation Parameter (VP) of anti-Candida species action of antibiotics against С. albicans and С. tropicalis (37-44%) was higher than in cases of antibiotic action against С. krusei and C. glabrata (30-35%). The growth of C. albicans or

С. tropicalis was inhibited by PBL in similar manner. aLB revealed stronger and prolonged effectiveness against Candida species.

Interaction between PBL and Candida species suspensions: Examples of differences of PBL interactions to non-albians Candida species are shown in Fig. 1. In contrast to monophase interaction between aLB and C. krusei, interaction between aLB and C. tropicalis was biphasic.

Among non-albicans Candida species tested, C. krusei strains revealed higher degree of interaction to PBL (Table 4). Effectiveness of PBL in discriminating Candida species s strains was changed during 24-72 h incubation. The most significant changes were within first 24 h incubation (especially) followed by next 24 h (Table 4). As a rule, upon 72 h incubation Candida clinical strain individuality was significantly decreased.

Discussion

The spectrum of PBL reactions against urogenital Candida clinical strains was similar to that in case of intestinal Candida clinical strains [10]. It seems higher and prolonged effectiveness of aLB interacting to Candida suspensions is supported by both preferential specificity of aLB to the fungal (phospho)mannans and increased stability to the surrounding hydrolases [7, 9]. Our results indicate that PBL acts as a cascade (in time and in space) involving earlier antibiotic-like action of acidic PBL.

It appears the system “PBL - Candida” is highly sensitive, and C. albicans clinical strains can serve as indicators of complex and prolonged events discriminating strains [10]. Similarly to our data, in comparison to C. albicans, C. glabrata and C. krusei are usually demonstrated the lower susceptibility to fluconazole and some other azoles, and reduced susceptibility to amphotericin B [11]. It is likely non-albicans Candida species strain differences established by us can be caused by increased level of hydrolases produced by C. tropicalis. Indeed, in contrast to C. krusei and

C. glabrata, the ability to produce increased levels of hydrolases allows to combine C. albicans (increased levels of phospholipases and acidic proteinases) and C. tropicalis (moderate levels of both types of hydrolases) into similar groups [12, 13]. It seems increasing interstrain similarity upon prolonged incubations was due to time-dependent increasing expression and production of hydrolases especially in case of C. tropicalis. Indeed, the direct correlation between Candida biomass growth and production of inducible hydrolases was demonstrated [12]. As a result, it is likely that PBL affinity to hydrolase-treated yeast cells must be changed.

The use of PBL allows classifications of responses of Candida species to antimicrobials. Earlier we classified such C. albicans responses to the presence of PBL-discs as: distant or not, border or internal, symmetrical or non-symmetrical, normal or stress [10]. The data presented above (Table 3) allow further grouping Candida species responses depending on sensitivity to azoles and non-azoles, preferential sensitivity to some azoles or their block.

The data of the Table 4 demonstrate quantitative sensitivity and reliability of using PBL (aLB, or aLL, or panel of both) in discrimination of non-albicans Candida species. Results indicate further basis for typing non-albicans Candida species clinical strains using combinations of LB and LL.

Conclusions: The data support a general statement that PBL act as universal regulators and signals in biotopes, and can serve as imitators of probiotics [5]. It seems the system “PBL – Candida clinical strains” can help in monitoring biotope “Human - Microbiota” status. PBL function as coupled to pathogen system (expression of pathogen virulence induces increasing antipathogen PBL activities). PBL may be used as effective ingredients of new system antimicrobial compositions. In this respect, anti-Candida species synergistic combinations of probiotic LB and LL, PBL and antibiotics are perspective.

References

1. Aleshkin V.A., Amerhanova A.M., Pospelova V.V., Afanasyev S.S. & Shenderov B.A. (2008) History, present situation, and prospects of probiotic research conducted in the G.N. Gabrichevsky Institute for Epidemiology and Microbiology. Microbial Ecolology in Health & Disease. 20: 113-115.

2. Lakhtin V.M., Afanasyev S.S., Alyoshkin V.A., Nesvizhsky Y.V., Pospelova V.V., Lakhtin M.V., Cherepanova Y.V. & Agapova Y.V. (2008) [Strategical aspects of constructing probiotics of the future (in russian)]. Vestnik Rossiiskoy Academii Meditsinskih Nauk. 2: 33-44, ISSN 0869-6047.

3. Lakhtin V., Lakhtin M. & Alyoshkin V. (2011) Lectins of living organisms. Anaerobe. 17: No 6, DOI:10.1016/j.anaerobe.2011.06.004.

4. Lakhtin V.M., Alyoshkin V.A., Lakhtin M.V., Afanasyev S.S., Pospelova V.V. & Shenderov B.A. (2006) [Lectins, adhesins and lectin-like substances of lactobacilli and bifidobacteria (in russian)]. Vestnik Rossiiskoy Academii Meditsinskih Nauk. 1: 28-34, ISSN 0869-6047.

5. Lakhtin M.V., Alyoshkin V.A., Lakhtin V.M., Nesvizhsky Y.V., Afanasyev S.S. & Pospelova V.V. (2010) [The role of lectins from probiotic microorganisms in sustaining the macroorganism (in russian)]. Vestnik Rossiiskoy Academii Meditsinskih Nauk. 2: 3-8, ISSN 0869-6047.

6. Lakhtin M., Lakhtin V., Alyoshkin V. & Afanasyev S. (2011) Lectins of beneficial microbes: system organization, functioning, and functional superfamily. Beneficial Microbes. 2: 155-165, DOI: 10.3920/BM2010.0014.

7. Lakhtin V.M., Lakhtin M.V., Pospelova V.V. & Shenderov B.A. (2006) Lactobacilli and bifidobacteria lectins as possible signal molecules regulating intra- and interpopulation bacteria-bacteria and host-bacteria relationships. Part I. Methods of bacterial lectin isolation, physicochemical characterization, and some biological activity investigation. Microbial Ecology in Health & Disease. 18: 55-60.

8. Lakhtin V.M., Lakhtin M.V., Pospelova V.V. & Shenderov B.A. (2007) Lectins of lactobacilli and bifidobacteria. II. Probiotic lectins of lactobacilli and bifidobacteria as possible signal molecules regulating inter- and intra-population relationships between bacteria and between bacteria and the host. Microbial Ecology in Health & Disease. 19: 153-157.

9. Lakhtin M.V., Lakhtin V.M., Alyoshkin V.A., Afanasyev S.S. & Alyoshkin A.V. (2010) [Lectins and enzymes in biology and medicine (in russian)]. Moscow: “Dynasty” Publishing House, 496 pp., ISBN 978-5-98125-076-7.

10. Lakhtin M.V., Alyoshkin V.A., Lakhtin V.M., Afanasyev S.S., Pozhalostina L.V. & Pospelova V.V. (2010) Probiotic lactobacillus and bifidobacterial lectins against Candida albicans and Staphylococcus aureus clinical strains: New class of pathogen biofilm destructors. Probiotics & Antimicrobial Proteins. 2: 186-196, DOI: 10. 1007/s12602-010-9046-3.

11. Majithiya J., Sharp A., Parmar A., Denning D.W. & Warn P.A. (2009) Efficacy of isavuconazole, voriconazole and fluconazole in temporarily neutropenic murine models of disseminated Candida tropicalis and Candida krusei. J Antimicrobial Chemotherapy. 63: 161–166.

12. Bramono K., Yamazaki M., Tsuboi R. & Ogawa H. (2006) Comparison of proteinase, lipase and α-glucosidase activities from the clinical isolates of Candida species. Japanese J. Infect. Dis. 59: 73-76.

13. Oksuz S., Sahin I., Yildirim M., Gulcan A., Yavuz T., Kaya D. & Koc A.N. (2007) Phospholipase and proteinase activities in different Candida species isolated from anatomically distinct sites of healthy adults. Japanese J Infect Dis. 60: 280-283.

14. Botina S.G. Auto-essay of the thesis of DSc (Biotechnology). Moscow, 2011.

15. Subbotina M.E. Auto-essay of the thesis of PhD (Genetics). Moscow, 2009.

Table 1. Probiotic lactobacillus and bifidobacterial strains used in the work

No	*Species, strains**	Previous names	Ingredients of Probiotics in Russia
1	L. helveticus NK1	L. acidophilus NK1	Acilact, Normospectrum, Polybacterin
2	L. casei/paracasei K₃III₂₄	L. acidophilus K₃III₂₄	Acilact, Normospectrum
3	L. helveticus 100_ash	L. acidophilus 100_ash	Acilact
4	B. longum MC-42	B. adolescentis MC-42	Bifidin
5	B. bifidum 1	B. bifidum 1	Bifidok, Bifidumbacterin

*[14, 15].

Table 2. Characteristics of some urogenital non-albicans Candida clinical strains used

Species	Strains	Lactobacilli in samples*	Bifidobacteria in samples
C. tropicalis	73	> 10⁵	Not
C. tropicalis	633	> 10⁴	Not
C. tropicalis	665	> 10⁴	Not
C. tropicalis	738	> 10⁴	Not
C. krusei	396	>10⁵	> 10³
C. krusei	584	Not	Not
C. krusei	660	>10⁵	> 10³
C. krusei	687	>10⁵	> 10³

* Cfu mL^-1

Table 3. Anti-Candida species action of antibiotics and probiotic bacterial lectins

Candida sp

Inhibition (diameter, mm)

A I K С N F aBL aLL

С. albicans

22.90 30.70 40.00 35.60 22.50 39.60 14.80 10.00

±4.04 ±6.89 ±4.47 ±5.78 ±6.01 ±3.50 ±1.79 ±2.00

C. krusei

19.75 29.43 29.14 30.25 19.50 25.50 10.00 15.50

±2.25 ±9.94 ±9.58 ±6.45 ±2.07 ±4.11 ±6.93 ±6.61

C. tropicalis

21.33 25.00 32.67 34.00 21.32 31.56 3 13.14 9.33

±1.41 ±4.54 ±8.18 ±3.87 ±1.39 ±9.76 ±4.30 ±2.31

Table 4. Interaction of probiotic bacterial lectins to С. tropicalis (I) or С. krusei (II) suspensions

Lectin

dilutions,

Candida sp

Bifidobacterial lectins

24 h 48 h

Lactobacillus lectins

24 h 48 h

1:10, I

36.43±4.50(7)* 67.14±8.41 (7)*

31.57±2.94(7)* 96.43±18.89(7)*

54.12±7.08(8) 62.12±7.68 (8)

34.60±2.51(5)* 64.43±12.11(7)*

26.60±0.89(5)* 38.00± 8.48(7)*

43.62±40.23(8) 60.12±52.17(8)

1:100, I

37.38±2.07(8)* 72.87± 7.36 (8)

33.50±2.98(8)* 80.75±21.40(8)

58.37±6.23(8) 69.00± 9.40 (8)

35.75±4.17(8) 52.86±14.18(7)*

35.00±0.76(8) 39.00±1.85(7)*

34.50±1.41(8) 42.86±12.70(8)

1:1000, I

36.63±2.92(8)* 61.87±3.98(8)*

25.00±1.60(8)* 46.12±3.91(8)*

37.87±1.88(8) 43.50±2.62 (8)

36.88±2.95(8)* 63.75± 6.36(8)*

33.25±1.39(8)* 38.00±1.85(8)*

33.50±1.51(8) 42.88±14.77(8)

1:10000, I

39.38±3.74(8)* 67.75±8.55(8)*

25.86±2.79(7)* 40.37±6.48(8)*

41.25±26.03(8) 45.37±24.87(8)

35.62±2.56(8) 71.17±8.77(6)*

35.00±1.93(8) 39.37±2.87(8)*

35.37±1.77(8) 44.75±13.62(8)

OD_600 nm in relative units. *The differences between I and II: P < 0.05 or P < 0.01. C= control.

Fig. 1. Influence of bifidobacterial lectins on yeast suspensions of non-albicans Candida species clinical strains upon 24 h co-incubation. Abscissa: Original suspensions (0.5 MacFarland) diluted to 10-10,000 times. Ordinate: D₆₀₀ in relative units (experimental data minus control data).