In vitro evaluation of anti-infective activity of a Lactobacillus plantarum strain against Salmonella enterica serovar Enteritidis

Abstract

Background: Salmonella enterica serovar Enteritidis infections are known to exhibit worldwide prevalence with increased morbidity and mortality. The conventional strategies like antibiotic therapy and vaccination have not only proved to be of sub-optimal efficacy but also led to the development of multidrug resistant strains of Salmonella. Antimicrobial activities of probiotics against various enteropathogens and other health promoting effects have assumed greater significance in recent years. The present study aims to evaluate the efficacy of a Lactobacillus plantarum strain (KSBT 56, isolated from a traditional food product of India), in preventing Salmonella enterica serovar Enteritidis growth and pathogenicity in vitro.

Methods and results: The cell free culture supernatant (CFCS) of KSBT 56 strain notably inhibited the growth of Salmonella Enteritidis without affecting the growth of other gram-positive lactic acid bacteria. The isolated KSBT 56 strain produces lactic acid similar to other standard probiotic strains like Lactobacillus plantarum MTCC 1407. The free radical production by KSBT 56 strain was studied by using sodC mutant of S. Enteritidis, which exhibited reduced growth in the presence of CFCS of the KSBT 56 strain, indicating the inhibitory activity of free radicals on the growth of S. Enteritidis. Our results also showed a significant reduction in the biofilm forming ability of Salmonella Enteritidis in the presence of the KSBT 56 strain (2 log cfu/ml, p = 0.01). Further, the anti-infective characteristics of KSBT 56 strain was validated by gentamicin protection assay which revealed 80% reduction in the invasion of Salmonella Enteritidis to HCT-116 cell line (Salmonella Enteritidis and KSBT 56 in a 1:1 ratio) and delayed addition of Salmonella Enteritidis by 1 h. Similarly, the reduced adhesion of Salmonella to the HCT-116 cells was observed along with the down regulation of hilA gene of Salmonella Pathogenicity Island 1 (SPI1) indicating that they might have acted synergistically to decrease the invasion of the pathogen into the cell line.

Conclusions: KSBT 56 strain effectively inhibited the growth, invasion and the biofilm forming ability of Salmonella Enteritidis without inhibiting the growth of other Lactobacillus strains. Overall, our result suggested that KSBT 56 can be used as a potential probiotic strain with considerable beneficial effects on the host.

Figure 1

Flow cytometer analysis of live/dead S. Enteritidis grown in the CFCS of KSBT 56 strain. S. Enteritidis expressing GFP are shown in Q3 in FITC channel. Propidium Iodide positive S. Enteritidis is shown in Q1 in PE-A channel. S. Enteritidis with compromised membrane expressing both GFP and propidium Iodide are seen in Q2. A. Untreated S. Enteritidis is shown in Q4. B.S. Enteritidis treated with 3% CFCS shows 86.6% of the population expressing GFP. C.S. Enteritidis treated with 5% CFCS has 87.8% of the population expressing GFP. D.S. Enteritidis treated with 7% CFCS shows 10.5% live S. Enteritidis expressing GFP. E.S. Enteritidis treated with 9% CFCS shows 1.08% live Salmonella in Q3 F.S. Enteritidis treated with 11% CFCS shows 0.1% live S. Enteritidis in the GFP-positive quadrant (Q3).

Figure 2

The growth of different Lactobacillus strain in the presence of CFCS of KSBT 56 strain. The absorbance of the cultures at 600 nm is plotted on the y-axis. The growth of different Lactobacillus strains was analysed by comparing absorbance at 0 h and 24 h of growth in presence of CFCS of KSBT 56.

Figure 3

Inhibition of growth of S. Enteritidis WT and ΔsodC mutant in the presence of CFCS (A) of KSBT 56 or live KSBT 56 (B). A.S. Enteritidis (SEn) WT or a mutant strain deficient of sodC gene (ΔsodC) were co-incubated with CFCS. B. The above groups were also co-incubated with live KSBT 56 bacterial culture. The cfu was enumerated by plating on LB agar plates supplemented with streptomycin. The presence of CFCS or KSBT 56 is indicated by (+) and the absence is indicated by (−). The growth of ΔsodC is compared with S. Enteritidis WT strain grown in the presence of CFCS or live KSBT 56 strain.

Figure 4

Inhibition of biofilm formation of S. Enteritidis by the KSBT 56 strain. The biofilm forming colonies of S. Enteritidis were enumerated on streptomycin LB Agar plates. The KSBT 56 bacterial culture was added to S. Enteritidis either simultaneously (0 h) indicated by (+) or at a time delay of 1 h. The absence of KSBT 56 is denoted by (−). KSBT 56 bacterial culture is plated on streptomycin LB Agar plates as control.

Figure 5

Effect of KSBT 56 on invasion of S. Enteritidis (A) and effect of CFCS of KSBT 56 on invasion of S. Enteritidis to HCT-116 cells. A. Gentamicin protection assay was performed to determine the invasion of S. Enteritidis into the HCT-116 cell line in the presence (+) or absence (−) of KSBT 56 strain. The pathogen and the KSBT 56 strain were either co-infected together into the cell line (0 h) or the pathogen was added at a time delay of (1 h). B. The effect of CFCS on invasion of S. Enteritidis was determined by co-incubating S. Enteritidis with the CFCS of KSBT 56 in 24- well tissue culture plate seeded with HCT-116 cell line. S. Enteritidis was also cultured with CFCS for 1 h before infection of HCT-116 cells. L. plantarum MTCC 1407 was taken as a reference strain. The invasion of S. Enteritidis to HCT-116 cells is taken as control.

Figure 6

Confocal images of Salmonella invasion (A-D) and Expression of hilA gene by RT-PCR (E). Confocal images were taken at 63X magnification using Leica CLSM. The membrane of HCT-116 cell lines were stained with plasma red dye and S. Enteritidis was tagged with GFP. The KSBT 56 strain was observed in phase contrast. A.S. Enteritidis invasion into HCT-116 in the absence of KSBT 56 strain. B.S. Enteritidis coinfected with KSBT 56 strain into HCT-116 cell line shows reduced invasion of S. Enteritidis. C. Delayed addition of S. Enteritidis after addition of KSBT 56 strain by 1 h further reduces the invasion of Salmonella into the cell lines. D. Merged image of panel C with phase contrast shows KSBT 56 adhering to HCT-116. E. RT-PCR analysis of hilA gene of S. Enteritidis grown in increasing concentration of CFCS of KSBT 56 strain. L. plantarum MTCC 1407 is a reference strain. The fold change in the expression of hilA gene is compared to S. Enteritidis WT (Untreated). SEn refers to S. Enteritidis and LP refers to L. plantarum MTCC 1407.