Synbiotic-like effect of linoleic acid overproducing Lactobacillus casei with berry phenolic extracts against pathogenesis of enterohemorrhagic Escherichia coli

To determine the effect of prebiotic like component BPEs, probiotic strain Lc or Lc + CLA or the metabolites produced by Lc or Lc + CLA in CFCSs on the growth inhibition of EHEC, we co-cultured EHEC with Lc or Lc + CLA or their CFCSs with different concentrations of BPEs [0.1 mg/ml gallic acid equivalent (GAE) or 0.5 mg/ml GAE or 1.0 mg/ml GAE]. In the presence of BPEs either of 0.1 mg/ml GAE or 0.5 mg/ml GAE, the growth of EHEC was not significantly reduced compared to the control but when the concentration of BPE was raised to 1.0 mg/ml GAE, the growth of EHEC was reduced significantly within 24 h (Fig. 1A–C). Further, either probiotic strain, Lc or Lc + CLA in combination with 0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE of BPEs intensified the reduction of the growth of EHEC in a time dependent manner but the inhibitory effect of Lc + CLA in presence of 1.0 mg/ml GAE was observed at maximum level [> 8.0 logs colony forming unit (CFU)/ml reduction] than the other treatments (Fig. 1C). CFCS collected from of Lc (CFCS-Lc) or Lc + CLA (CFCS-Lc + CLA) in the presence of BPEs of different concentrations also showed inhibitory effects on the growth of EHEC (Fig. 1A–C). After 24 h, the reduction in the growth of EHEC was observed ranging from < 1.0 log CFU/ml (CFCS-Lc in presence of 0.1 mg/ml GAE of BPE) to > 3.0 logs CFU/ml (CFCS-Lc + CLA in presence of 1.0 mg/ml GAE of BPE). After 48 h of incubation, the growth of EHEC was reduced more than 3.0 logs CFU/ml by CFCS-Lc in presence of 1.0 mg/ml GAE of BPEs and more than 4 logs CFU/ml by CFCS-Lc + CLA in presence of BPEs of 1.0 mg/ml GAE concentration compared to control group (no CFCS or BPEs added to the growth medium). After 72 h of incubation, the most effective growth reduction of EHEC was observed by CFCS-Lc + CLA in presence of 1.0 mg/ml GAE of BPEs with no detectable growth (Fig. 1C).

The adhesion ability of EHEC to INT-407 cells in the presence of different concentrations (0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE) of BPEs, in combination with Lc or Lc + CLA itself or their metabolites containing CFCSs (CFCS-Lc and CFCS-Lc + CLA) was reduced significantly from 0.5 log CFU/ml to 4.0 logs CFU/ml (Fig. 2A–C). BPEs (0.1 mg/ml GAE or 0.5 mg/ml GAE) without the probiotics or their CFCSs, numerically reduced the adhesion ability, though 1.0 mg/ml GAE reduced adhesion ability around 0.6 log CFU/ml, significantly (Fig. 2A–C) when compared to the control (only medium without any treatments).

We observed that pre-treatments of EHEC with all the treatments accept 0.1 mg/ml GAE of BPE, significantly increased the percentage of injured bacterial cells ranging from 36.33 to 58.97% when compared to control (without any treatments in growth media) (Table 1). The auto-aggregation capacity of EHEC decreased significantly by CFCS-Lc in presence of 1.0 mg/ml GAE of BPE or CFCS-Lc + CLA in presence of 0.5 mg/ml GAE or 1.0 mg/ml GAE of BPEs, and CFCS-Lc + CLA in presence of 1.0 mg/ml GAE of BPE was the most effective when compared to control group. The auto-aggregation capacity of EHEC was reduced numerically by 0.1 or 0.5 or 1.0 mg/ml GAE of BPE, CFCS-Lc in presence of 0.1 mg/ml GAE or 0.5 mg/ml GAE of BPE, CFCS-Lc + CLA in presence of 0.1 mg/ml of GAE but not significantly (Table 1). We also found that cell surface hydrophobicity of EHEC was reduced significantly with all the pre-treatments ranging from 4.65 to 1.45% accept 0.1 mg/ml GAE of BPE. Also, BPE of 0.1 mg/ml GAE concentration reduced the cell surface hydrophobicity of EHEC to 13.1% when compared to control group but only numerically (Table 1).

Biofilm formation is the process of attachment to a surface which can affect the growth rate and gene transcription ability of a bacteria and when EHEC was incubated in presence of BPEs (0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE) without CFCSs (collected from overnight culture of Lc or Lc + CLA) the biofilm formation ability was reduced significantly around 1.0 log CFU/ml. BPEs in combination with CFCS-Lc or CFCS-Lc + CLA also reduced the biofilm formation ability of EHEC when compared to the control group (growth media without treatment), significantly from < 1.0 log CFU/ml to > 4.4 logs CFU/ml and CFCS-Lc + CLA was more effective in the reduction of the biofilm formation in presence of same concentration of BPE in comparison with CFCS-Lc (Fig. 3A).

Expression of espA gene of EHEC, which is responsible for encoding secreted protein related to signal transduction leading to A/E lesion formation, was significantly down-regulated at a range from 1.2- to 62.5-folds by either CFCS-Lc or CFCS-Lc + CLA collected from Lc or Lc + CLA in the presence of different concentrations of BPEs (Fig. 4a–c). The expression level of espB and espD genes which are also involved in signal transduction leading to A/E lesion formation were down regulated significantly ranging from 1.1- to 28-folds in the treatment assay. The expression level of eaeA encoding intimin protein required for intimate attachment was also down regulated significantly more than 1.1-folds to more than 3.9-folds in the assay. The expression level of locus of enterocyte effacement (LEE)-encoded regulator, ler was downregulated significantly > 1.3- to > 8.5-folds by all the concentrations of BPEs with or without CFCS-Lc or CFCS-Lc + CLA. The expression level of tir responsible for translocated intimin receptor was also downregulated around onefold by different concentrations of BPEs with or without CFCS-Lc or CFCS-Lc + CLA (Fig. 4a–c).

In this study, enterohemorrhagic Escherichia coli EDL933 (EHEC) (ATCC 700927) was grown on Luria-Bertani (LB) agar (EMD Chemicals Inc., USA) for 18 h at 37 °C under aerobic conditions (Thermo Fisher Scientific Inc., USA). Probiotic strains, Lactobacillus casei (Lc) (ATCC 334) and lineolate over-expressed bioactive L. casei (Lc + CLA) [23, 24] were grown on de Man Rogosa Sharpe (MRS) agar (EMD Chemicals Inc., USA) overnight at 37 °C under aerobic condition with 5% CO₂ (Thermo Fisher Scientific Inc., USA).

Commercial blackberry and blueberry pomaces was donated by Milne Fruit Products Inc., Prosser, WA, USA, and BPE was extracted following the protocol previously reported [13]. Spectrophotometric method was used to measure the concentration of BPE and expressed as GAE [43]. BPE was comprised of blackberry and blueberry pomace extracts at 1:1 v/v ratio for this study.

Human intestinal epithelial (INT-407) (ATCC CCL-6) cells were cultured at 37 °C, standard condition (5% CO₂) in Dulbecco’s Modified Eagle Medium (DMEM) (Corning Cellgro, USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Corning Cellgro, USA) and 50 µg/ml of gentamycin (Lonza, USA). For monolayer preparation, INT-407 cells were seeded in 24-well culture plate (Greiner Bio-one Inc., USA) at 2 × 10⁵ cells/ml and maintained following the standard protocol described as above to form > 90% confluence monolayer. Before use, the monolayers were washed with phosphate buffer saline (PBS) three times and immersed in antibiotic free DMEM supplemented with 5% heat-inactivated FBS [24].

Cell free culture supernatant (CFCS) of overnight liquid cultures of Lc, and Lc + CLA were collected following the method previously reported by our laboratory [21] and collected CFCSs were filtered and stored at 4 °C.

Inhibition of EHEC growth was carried out in presence of different concentrations of BPEs (0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE) and/or Lc or LC + CLA. All bacterial strains were grown on selective respective agar plates following the method described above. A volume of 400 µl of Lc or Lc + CLA bacterial suspension containing approximately 10⁷ CFU/ml was mixed with equal volume of EHEC suspension containing approximately 10⁶ CFU/ml in 3.2 ml of LB broth and incubated at 37 °C in presence of different concentrations (0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE) of BPE. Serial dilutions were performed in PBS, followed by plating on LB agar for EHEC at 0, 24, 48, and 72 h time points. For inhibition assay with CFCSs, instead of live Lc or Lc + CLA, 400 µl of CFCS-LC (collected from Lc) or CFCS-Lc + CLA (collected from Lc + CLA) with/without BPEs were mixed with equal volume of EHEC suspension containing approximately 10⁶ CFU/ml in 3.2 ml of LB broth and incubated at 37 °C and counted following the above mentioned plating methods at 0, 24, 48 h and 72 h time points.

Adhesion of EHEC to INT-407 cell was performed following the method described previously [21, 24]. Briefly, INT-407 cell monolayers were pre-treated with 100 µl DMEM (control), different concentrations of BPEs (0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE) or with probiotics or their CFCS in presence of different concentrations of BPEs (0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE) for 1 h (triplicates). After pre-treatment, 100 µl of EHEC, bacterial suspension with multiplicity of infection (MOI) of 10 (2 × 10⁶ CFU/ml) were inoculated into each well, followed by 2 h incubation, lysis by 0.1% Triton X-100 for 15 min, serial dilutions and plating for quantification.

Changes of physicochemical properties including cell surface hydrophobicity, auto-aggregation and injured cell ratio of EHEC were evaluated following the methodologies previously described [16] with modifications in culture condition. In brief, EHEC was grown in LB broth or LB broth with BPEs (0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE) or CFCSs collected from Lc or Lc + CLA in combination with BPEs at 37 °C for 18 h.

The ability of EHEC to form biofilms on glass surfaces in the absence or presence of CFCSs and/or BPEs was performed following the method previously described [16]. Briefly, 100 µl of EHEC, containing approximately 5 × 10⁵ CFU/ml, was inoculated in triplicate in wells of 6-well plates (Corning, USA) containing 22 × 22 mm² glass slides. Wells containing LB broth (control) or LB broth supplemented with different concentrations of BPEs (0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE), or CFCSs with BPEs were incubated for 48 without shaking at 37 °C. Then, the glass slides were rinsed with PBS for five times and bacterial cells were recovered using sterile cell scraper (VWR, USA) from the glass surface and enumerated.

EHEC was grown in the absence or presence of BPEs (0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE) or/and CFCSs collected from Lc or Lc + CLA and RNA was extracted according to the protocol of ZR Bacterial RNA MiniPrep kit (Zymo Research Corp., USA). The RNA quantification was carried out using NanoDrop spectrophotometer (Thermo Scientific Inc., USA) and cDNA synthesis was performed according to the protocol provided by Quanta Biosciences, USA. The custom-synthesized oligonucleotide primers for espA, espB, espD, eaeA, ler, tir of EHEC were purchased from Eurofins MWG Operon, USA and methodology previously described [44] was followed to perform the qRT-PCR assay.

Collected data were analyzed by the Statistical Analysis System software (SAS Institute Inc., USA). The one-way analysis of variance (ANOVA) for each single time point followed by Tukey’s test was used to evaluate the various treatments and significant differences among control and treatments were determined based on significant level of 0.05.