Lactobacillus species mediated synthesis of silver nanoparticles and their antibacterial activity against opportunistic pathogens in vitro

Bacterial strains and synthesis of silver NPs

The capacity to precipitate silver and synthetize NPs was evaluated for 22 different strains (Table 1). Bacteria were cultured in the MRS medium at 37°C for 24 h. After incubation, the bacterial biomass was harvested by centrifugation at 1.500 g for 10 min and washed several times with sterile deionized water. For the synthesis of Ag NPs, 1 mM AgNO₃ was added to bacterial pellet to give an optical density of 1.0 at 630 nm.

Characterization of silver NPs

The UV–Vis spectrum was determined using a SPECORD M-400 UV-Vis spectrophotometer (Japan). The absorbance was measured in the range of 400-800 nm. Deionized water was used as the blank.

Transmission electron microscopy (TEM) measurements were performed using a JEOL JEM 1400 instrument operated at an accelerating voltage of 80 kV. Size of the NPs was determined using TEM micrographs. A total of 100 particles were evaluated for each strain.

Capsular exopolysaccharides isolation

For isolation of capsular exopolysaccharide (CPS), lactobacilli strains were grown in MRS medium (1000 mL) at 37°C for 24 h. The cultures were centrifuged at 12 000 g for 15 min at 4°C and the cell pellets were collected for the extraction of CPS. The cells were washed with saline solution and further treated with 0.5% phenol for 4 h at room temperature.⁷ After centrifugation, to remove the cells, the supernatant was precipitated with three-fold (v/v) of chilled ethanol. After standing overnight at 4°C, the precipitate was collected by centrifugation (12 000 g, 20 min at 4°C), suspended in 10 mL of distilled water, and dialyzed against 3 L of distilled water for 2 days with 3 water changes per day. The dialysate was lyophilized and the total sugar concentration was determined by the phenol-sulfuric method, using glucose as a standard.

Monosaccharide composition of capsular exopolysaccharides

The CPS samples were hydrolyzed with 1 mL of 2 M trifluoroacetic acid (TFA) at 100°C for 6 h. TFA was removed by vacuum evaporation. A volume of 0.3 mL derivatization reagent (32 mg hydroxylamine hydrochloride mL-¹, 40 mg 4-dimethylaminopyridine mL-¹ in pyridine/methanol [4:1 v/v]) was added to each dried sample, and then the samples were incubated at 75°C for 25 min. After incubation, the tubes were cooled down to the room temperature and 0.3 mL of acetic anhydride was subsequently added and incubated at 75°C for 25 min. To the reaction mixture, 1 mL of dichloroethane was added. Excess reagents were removed by double extraction using 1M hydrochloric acid and followed by H₂O washing (×3) steps. Dichloroethane phase was removed, dried, and redissolved in 300 μL of heptane/methyl acetate mixture (1:1 v/v).⁸

The GC/MS analysis of acetylated aldononitriles was carried out using Agillent 6890N/5973inert (Agillent Technologies, Inc., Richardson, TX USA). A capillary column НР-5MS (30 m ×0.25 mm (i.d), film thickness 0.25 μm) (J&W Scientific, USA) was used. The temperatures of injector and MS interface were 250°С and 280°С, respectively. The oven start temperature, 160°C, was held for 8 min, ramped up to 240°C at the rate of 5°C/min, and held for 6 min. The injection volume was 1 μL, and the split ratio was 1:50. For detection, the EI of samples were scanned in the range of 38-5400 m/z. Helium was used as mobile phase (flow rate of 1.2 mL min-¹). Derivatised monoschacarides were identified by comparing their retention times with standard mixtures of monosacharides (galactose, mannose, xylose, glucose, fructose, arabinose, rhamnose) and their mass fragmentation spectra with NIST02 MS library.

Antimicrobial activity of silver NPs

The antimicrobial action was determined by agar well diffusion assay. Antibacterial action was tested for synthesized Ag NPs and cell free supernatant against opportunistic pathogens of Gram-positive bacteria (Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, Bacillus cereus ATCC 11778), Gram-negative bacteria (Pseudomonas aeruginosa ATCC 9027, Proteus vulgaris ATCC 6896, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 10031, Salmonella enterica NCTC 6017, Shigella sonnei GICK 337, S. flexneri GICK 233169), and yeast (Candida albicans UCM Y-1918) on Mueller–Hinton (MH) agar plates. Agar wells of 6 mm diameter were prepared and loaded with 50 μL of Ag NPs solution and 50 μL of bacterial cell suspension without AgNO₃ as control. The plates were incubated at 37°C for 24 h and the diameter of zone of inhibition was measured as indicated by clear area devoid of growth of test-strains. Each experiment was done twice.

Statistical analysis

A one-way analysis of variance (ANOVA) was performed using Statistica Software version 7.0. Mean values were compared using LSD test (least-significant difference, p<0.05) and different letters were used to label values with statically significant differences among them.

Results and discussion

Silver NPs synthesis

Only ten from 22 of Lactobacillus strains had the potential to reduce the silver ions to silver NPs (Table 1). The white bacterial biomass of these strains changed to yellowish brown after incubation with 1 mM aqueous AgNO₃ solution suggesting the formation of silver NPs. The UV-VIS absorption spectra of all bacterial suspensions were monitored and a strong peak in the visible region typical for the Ag NPs (Fig. 1A) was obtained only for 10 bacterial cell suspensions.

Fig. 1. A- UV/VIS absorption spectra of the Ag NPs prepared using L. plantarum 92T cell suspension. B- Transmission of electron micrographs of Ag NPs obtained with lactobacilli after 24 h of incubation in AgNO₃ solution. (a) – cell of strain L. fermentum 215 with Ag NPs on the surface (scale bar corresponds to 500 nm); (b, c) – accumulation of Ag NPs in capsular polymers of L. casei strain (scale bar corresponds to 100 nm).

× A- UV/VIS absorption spectra of the Ag NPs prepared using L. plantarum 92T cell suspension. B- Transmission of electron micrographs of Ag NPs obtained with lactobacilli after 24 h of incubation in AgNO₃ solution. (a) – cell of strain L. fermentum 215 with Ag NPs on the surface (scale bar corresponds to 500 nm); (b, c) – accumulation of Ag NPs in capsular polymers of L. casei strain (scale bar corresponds to 100 nm).

Fig. 1.

The results were confirmed with the TEM analysis of the samples (Fig 1B), which showed the presence of spherical nano-sized particles in the same 10 samples (Table 1). Thus, the ability to reduce the silver ions was strain-specific and was not dependent to the Lactobacillus species. In most cases Ag NPs had a diameter between 10 and 40 nm (89.6%), whereas 0.7 % of Ag NPs had a diameter smaller than 10 nm and 9.7% of NPs were greater than 40 nm. Therefore, the NPs in this work (Table 1) were in the same approximate range of 5–60 nm reported by authors for Ag NPs produced from LAB and other microorganisms.^6,9 The Ag NPs produced by L. rhamnosus CCM 1825^T and L. plantarum 92T had the smallest mean particle size. The NPs with the largest size were synthesized by strain L. acidophilus NCS. The particle size distribution in case of L. casei CCM 7088^T was larger than that in case of other Lactobacillus strains. The narrowest size distribution was in case of strains L. plantarum 92T and L. fermentum CCM 7192^T.

Capsular exopolysaccharides analysis

We investigated the CPS of five Lactobacillus strains that produced NPs with different size. The CPSs of two strains that did not produce Ag NPs were investigated for comparison. The monosaccharide composition of CPS from different Lactobacillus strains varied significantly (Table 2). Most of them were composed of galactose, glucose, mannose, and rhamnose in different molar ratios. The capsular exopolysaccharide of L. plantarum 11/16 contains only glucose and galactose. This could explain the ability to reduce the silver ions and produce nanoparticle and its different sizes and size distribution. As shown by other authors,¹⁰ the difference in the structure of monosaccharides and disaccharides influences the Ag NPs size.

Antimicrobial action of silver NPs

Antimicrobial properties of Ag NPs from two Lactobacillus strains was studied (Fig. 2). The highest antimicrobial activity was observed against Candida albicans, followed by S. epidermidis and S. aureus. It was found that Ag NPs with large size (30.65±5.81 nm) obtained from L. acidophilus 58p were more active against S. epidermidis, E. coli, K. pneumonia, S. flexneri, and S. sonnei compared with Ag NPs from L. plantarum 92T (19.92±3.4 nm) (p<0.05) (Fig. 2).

Fig. 2. Size of the inhibition zone for Ag NPs synthesized by L. plantarum 92T and L. acidophilus 58p strains against the tested opportunistic pathogens. * Indicates a significant difference (p < 0.05) in the size of inhibition zones.

× Size of the inhibition zone for Ag NPs synthesized by L. plantarum 92T and L. acidophilus 58p strains against the tested opportunistic pathogens. * Indicates a significant difference (p < 0.05) in the size of inhibition zones.

Fig. 2.

Antibacterial activity of Ag NPs depends on the reducing agents and stabilizers used for synthesis.^3,11 Smaller particles having the larger surface area available for interaction would give more bactericidal effect than the larger particles.³ As was shown by Panacek et al.¹⁰ Ag NPs synthesized using disaccharides have a higher antibacterial activity than those synthesized using monosaccharides. The sizes of the colloidal Ag particles were smaller for disaccharide than for monosaccharide and thus may be responsible for the observed antibacterial activity. However, there was some exceptions, for example, galactose had the largest Ag NPs (50 nm), and had higher antibacterial activity against methicillin-susceptible S. epidermidis than those synthesized using glucose (44 nm). In addition, there were equal MIC values for NPs with different sizes; for example, MIC of Ag NPs synthesized via reduction by glucose (44 nm), maltosa (25 nm), and lactose (35 nm) against S. aureus, was 6.75 μg/mL.⁸ Thus, the size, size distribution, and differences in the antibacterial activity of Ag NPs produced by L. plantarum 92T and L. acidophilus 58p strains may be due to their capsular biopolymers composition, but further work is required to characterize these differences and other biological activities of synthesized Ag NPs.

At the present work, Ag NPs are extracellular-synthesized by the biomass of the Lactobacillus strains of different species. Therefore, the components produced by these strains, causing the absorption and reduction of silver ions to Ag NPs are most likely associated with the surface of bacterial cells. This may indicate the location of Ag NPs on the surface of cells, preferably in the capsular layer (Fig. 1B). Application of the LAB for the synthesis of Ag NPs has already been reported, and the ability to accumulate Ag NPs on their surfaces and inside the cells has been reported.¹² However, the exact reaction mechanism leading to the formation of Ag NPs by LAB needs to be elucidated.