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Remediation of intramacrophageal Shigella dysenteriae type 1 by probiotic lactobacilli isolated from human infants' stool samples
Reprint requests: Dr. Rupinder Tewari, Department of Microbial Biotechnology, Panjab University, Sector 14, Chandigarh 160 014, India e-mail: rupinder@pu.ac.in
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Abstract
Background & objectives:
Shigella dysenteriae is one of the most virulent pathogens causing bacillary dysentery and is responsible for high mortality in infants. To reduce the load of antibiotic therapy for treating shigellosis, this study was carried out to assess the ex vivo effect of novel probiotic lactobacilli, isolated from infant's stool samples, on killing S. dysenteriae type 1 residing in the rat macrophages.
Methods:
Stool samples from infants were collected, processed for the isolation of lactobacilli and screened for the probiotic attributes (acid tolerance, bile tolerance, ability to adhere intestinal cells and anti-S. dysenteriae activity). The effect of cell-free supernatant of lactobacilli on Shigella- infected macrophages in terms of phagocytic ability, extent of lipid peroxidation, nitrite, superoxide dismutase and glutathione levels was evaluated.
Results:
Based on the probiotic attributes, three lactobacilli were isolated from the stool samples of infants. Using classical and molecular tools, these isolates were identified as Lactobacillus pentosus, L. Paraplantarum and L. rhamnosus. All the three lactobacilli had the ability to kill intramacrophage S. dysentriae type 1. The anti-Shigella activity of the probiotic lactobacilli was attributed to increased antioxidative ability and decreased free radical production by the infected macrophages.
Interpretation & conclusions:
Probiotic cocktail of L. pentosus, L. paraplantarum and L. rhamnosus showed ex vivo killing of S. dysenteriae residing inside the rat macrophages significantly. This cocktail has the potential to be used as a natural alternative for treating S. dysenteriae infection, especially in infants, however, further studies need to be done to confirm these finding in vivo.
Keywords
Infant
Lactobacillus paraplantarum
Lactobacillus pentosus
Lactobacillus rhamnosus
macrophage
probiotic
Shigella dysenteriae
stool
Shigella being facultative intracellular pathogen, having prominent specificity for primate hosts mainly humans, is responsible for acute gastrointestinal infection named shigellosis1. Genus Shigella consists of four species namely S. boydii, S. dysenteriae, S. flexneri and S. sonnei. Amongst these four species, S. dysenteriae and S. flexneri are prominently found in developing world that can lead to deadly epidemics2. Moreover, S. dysenteriae type 1 attracts special attention due to its imperative property to produce potent enterotoxin – Shiga toxin that contributes to high attack rate, high case fatality rate and serious complications1. The situation becomes more serious because of lack of vaccine counterparts against S. dysenteriae type 1 and emergence of drug resistance to commonly used fluoroquinolones and aminoglycosides which was found to be more common in S. dysenteriae as compared to S. flexneri3.
One of the natural alternative ways to combat S. dysenteriae type 1-mediated infections is the use of probiotics4. Probiotics are live microorganisms that, when administered in adequate amounts, provide health benefits to the host5. In the past decade, the role of probiotics in controlling bacterial infections is on the rise5. However, only a limited number of reports on the use of probiotics for the control of S. dysenteriae are available. Only two reports46 have discussed mechanism behind lactobacilli-mediated killing of Shigella to a certain extent.
The present study was thus undertaken to isolate lactobacilli from stool samples of infants and check their probiotic and anti-Shigella activity. The main focus was on the treatment of S. dysenteriae-infected rat macrophages with probiotic lactobacilli isolated from infants' stool.
Material & Methods
This study was carried out in the department of Microbial Biotechnology, Panjab University, Chandigarh, India, from October 2011 to October 2013. Fresh stool samples were collected from healthy, breastfed infants (1-3 months old) attending vaccine clinic in the department of Pediatrics, Government Medical College, Chandigarh, and were subsequently processed. These samples were serially diluted in phosphate-buffered saline (pH 7.0) (HiMedia, Mumbai) and pour plated on de Mann Roger Sharpe-Bromocresol purple (MRS-BCP) (HiMedia) agar plates. Following incubation, the acid-producing colonies obtained on the plates were Gram stained and biochemically tested. These were subjected to Lactobacillus genus-specific polymerase chain reaction (PCR) with specific primers (Lacto F, Lacto R) for 16S-23S rRNA intergenic fragment of Lactobacillus genus7.
Anti-S. dysenteriae activity of lactobacilli isolates by agar well diffusion
Lactobacilli isolates were screened for anti-Shigella activity against S. dysenteriae type 1 (provided by the department of Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh). Anti-Shigella activity was checked by performing agar well diffusion assay8 on tryptone glucose extract agar plates (HiMedia).
Probiotic attributes of anti-S. dysenteriae lactobacilli
Anti-Shigella lactobacilli were further screened for acid (p H 2.0) tolerance for two hours and bile salts (oxgall, 1.5%) (HiMedia) tolerance for four hours, respectively9. Hydrophobicity index of isolates was calculated with xylene, higher the hydrophobicity of isolates greater is their adherence to intestinal cells9. Lactobacilli isolates showing hydrophobicity more than 30 per cent were subjected to adherence assay on cultured Caco-2 cells as bacterial cultures showing mean adherence to xylene more than 30 per cent were considered as hydrophobic10. The adhesion scores were calculated based on the number of bacteria enumerated from 20 microscopic fields. Percentage adhesion of lactobacilli isolates was calculated according to the following Formula11:
Biocompatibility amongst the selected isolates was checked on MRS agar plates. Isolates were streaked in line at a distance of 1 mm, perpendicular to each other on MRS agar plates and incubated for 24 h. After incubation, MRS agar plates were checked for growth of lactobacilli isolates.
Molecular identification of isolated anti-Shigella probiotic lactobacilli
Lactobacilli isolates showing probiotic attributes were subjected to molecular identification by performing 16S rRNA gene sequencing with primers 27f, 685r, 926f and 1492r12. BigDye terminator cycle sequencing kit (Applied Biosystems, USA) was used to sequence purified 16S rRNA gene fragment in 3130X Genetic Analyzer (Applied Biosystems). Sequence data obtained were analyzed by DNA sequences assembling software SEQUENCHER™ 4.10.1 (Gene Codes Corporation, Michigan, USA). Related sequences were determined from nucleotide database of National Centre of Biotechnological Information (NCBI). Alignment of all acquired and related sequences was done with Clustal W software (EMBL-EBL, http://www.ebi.ac.uk/Tools/msa/clustalw2/) using neighbour-joining method in accordance with MEGA4 and Kimura 2-parameter model (www.megasoftware.net) to construct phylogenetic tree. 16S rRNA gene sequences of isolated lactobacilli RT16-2, RT4-54 and RT27-17 were submitted to Bankit NCBI under GenBank accession numbers KJ802480, KJ802481 and KJ802482.
Ex vivo study of lactobacilli (individually and cocktail) against S. dysenteriae type 1
The anti-Shigella probiotic lactobacilli selected were grown for 48 h in MRS broth. The culture obtained was centrifuged at 2190×g for 10 min (Sigma 2-16 K, Newtown, Shropshire, UK) and lactobacilli cell-free supernatant (LCFS) was collected and filter sterilized using 0.22 μm filters. LCFS was analyzed for the presence of bacteriocin, lactic acid and hydrogen peroxide. Lactic acid content was determined in CFS supernatant13. Amount of hydrogen peroxide produced in CFS was also estimated14. CFS was treated with 1N sodium hydroxide till LCFS attained p H 7.0 and catalase (1 mg/ml) (HiMedia) for 60 min to eliminate anti-Shigella effects of lactic acid and hydrogen peroxide and treated supernatant was designated as S1. Further, CFS was subjected to protein digestion with enzymes pepsin, trypsin, chymotrypsin and renin (HiMedia) at a concentration of 1 mg/ml each for 60 min at 37°C to assure the presence of bacteriocin and designated as CFS S2. Treated LCFS (S1 and S2) was subjected to agar well diffusion assay to check for anti-Shigella activity.
Extraction of peritoneal macrophages from rat
Healthy Wistar rats (200-250 g) were procured from Central Animal Facility of Panjab University, Chandigarh. Rat peritoneal macrophages were isolated in accordance with method described by Chanana et al15. The study protocol was approved by the Institutional Animal Ethics Committee.
Intracellular killing of S. dysenteriae type 1 by macrophages
Rat peritoneal macrophages were infected with S. dysenteriae at 1:100 multiplicity of infection. Macrophage suspension (106 cells/ml), normal rat serum and infected S. dysenteriae suspension (108 cfu/ml) were mixed by vortexing and incubated for two hours at 37°C in five per cent CO2 incubator (4141, Forma™ series water jacketed, Thermo Scientific, USA). The suspension was further treated with CFS of lactobacilli individually, CFS cocktail of two lactobacilli and CFS cocktail of all the three lactobacilli taken together16 as mentioned in Table I.
Effect of LCFS (individually and cocktail) on macrophage functions
The macrophage suspensions were incubated for 18 h at 37°C in CO2 incubator and lysed with lysis buffer (20 mM Tris hydrochloride, 150 mM sodium chloride, 1 mM EDTA, 1 % triton-X-100 and 1 mM PMSF; HiMedia) in the ratio of 1:1 (v/v) and incubated for 20 min at 4°C. This reaction mixture was centrifuged at 101×g for 15 min and supernatant obtained was collected for assaying macrophage functions.
Estimation of lipid peroxidation (LPO)
Quantitative assessment of lipid peroxidation (LPO) in macrophage culture supernatant was carried out17. Protein content was determined by Bradford method18. Results were expressed in nanomoles of malondialdehyde (MDA) per milligramme of protein, using chromophore molar extinction coefficient as 1.56×105 M -1 cm -1.
Measurement of nitrite concentration
Nitrite [as an indicator of nitric oxide (NO)] content in macrophage culture supernatant was determined19. Quantification of nitrite was done through a standard curve of sodium nitrite (HiMedia).
Measurement of superoxide dismutase (SOD) activity
Method described by Kono20 was used to assay the levels of superoxide dismutase (SOD) in macrophage culture supernatant. Activity of SOD was expressed as units of SOD per milligram of protein in culture supernatant.
Reduced glutathione (GSH) levels
Glutathione (GSH) levels in macrophage culture supernatant were determined21. GSH levels were expressed in micromoles of GSH per milligramme of protein applying the molar extinction coefficient for chromophore as 13600/ M/cm.
Statistical analysis
Treatment group variations in comparison to positive and negative controls were determined by student t test using GraphPad statistical software (GraphPad Prism 4 software, La Jolla, USA).
Results
Based on the colony morphology (flat, creamish in colour, 2-3 mm colony, convex, raised circular form with entire margin) on MRS-BCP agar plates, 112 putative lactobacilli were isolated from 50 stool samples of infants. On further characterization involving Gram character (Gram-positive), shape (rods) and biochemical tests (catalase negative, triple sugar iron test showing acid production in slant and butt and lactose fermentation), 102 isolates were found to belong to the genus Lactobacillus. Using PCR involving amplification of 250 base pair (bp) Lactobacillus genus-specific 16S-23S RNA gene fragment, 99 isolates were confirmed as lactobacilli. The complete identification of the three selected probiotic lactobacilli was carried out on the basis of 16S rRNA gene sequencing. The organisms were identified as L. pentosus, L. paraplantarum and L. rhamnosus (GenBank accession numbers KJ802480, KJ802481 and KJ802482). The phylogenetic tree showing genetic relatedness amongst these isolates showed L. pentosus and L. paraplantarum closely related to each other and relatively distinct from L. rhamnosus.
Anti-S. dysenteriae type 1 activity and probiotic attributes
Only 26 of 99 isolates exhibited significant anti-S. dysenteriae activity (zone of inhibition ≥5 mm). The anti-Shigella lactobacilli were evaluated for probiotic attributes i.e., acid tolerance, bile tolerance, hydrophobicity and adherence to Caco-2 cells. Based on the results obtained, three anti-Shigella lactobacilli, namely, L. pentosus (RT4-54), L. paraplantarum (RT16-2) and L. rhamnosus (RT27-17) were selected for further studies. These showed 100 per cent bile tolerance at 1.5 per cent concentration of oxgall, high acid tolerance (92-96%) at p H 2.0, substantial hydrophobicity (41-63%) and adherence (15-26%) on Caco-2 cells (Table II). In addition, selected lactobacilli were completely biocompatible to each other.
Presence of anti-Shigella factors in LCFS
The maximum production of lactic acid was shown by L. rhamnosus (176±12.16 mg/ml) followed by L. pentosus (122.6±14.1 mg/ml) and L. paraplantarum (95±13.22 mg/ml). However, H2O2 production was maximum in case of L. paraplantarum (89.3±4.04 μg/ml), followed by L. rhamnosus (71.33±10 μg/ml) and L. pentosus (63±8.1 μg/ml). The treatment of LCFS of three probiotic lactobacilli with various proteases (pepsin, trypsin, chymotrypsin and renin) did not result in a change in the anti-Shigella activity as compared to the untreated LCFS, thereby suggesting a little role of the bacteriocins in anti-Shigella activity (Table III).
Effect of LCFS on intracellular killing of S. dysenteriae type 1
The results of the intracellular killing of S. dysenteriae type 1 in rat peritoneal macrophages with LCFS of probiotic lactobacilli are mentioned in Fig. 1. The range of Shigella killing using LCFS of individual lactobacilli was found to be 50-55 per cent (P <0.05) whereas intracellular killing of Shigella in the control group was 43.4±2.16 per cent. In another set of experiments, in which LCFS of two of the three was administered to the macrophages, the intracellular killing of Shigella increased up to 75-80 per cent (P <0.01). However, the maximum killing of Shigella (90.16±2.55%, P <0.01) was observed using a cocktail of all the three organisms.
- Effect of lactobacilli cell-free supernatant (LCFS) of probiotic lactobacilli on mean percentage of intracellular killing of Shigella dysenteriae type 1 by rat peritoneal macrophages. Values are expressed as mean±standard deviation of three individual values. P*< 0.05, **< 0.01, ***< 0.001 compared to killing of S. dysenteriae by untreated macrophages. C, control; T, treatment group.
Estimation of lipid peroxidation (LPO) and nitrite (NO)
To determine the level of damage to macrophage cell membrane during infection, the magnitude of LPO was measured in terms of level of MDA released by infected macrophage. The challenge group C2 (comprising macrophages and S. dysenteriae) showed 279.667±22.81 nanomoles of MDA/mg protein, which was significantly (P <0.05) elevated in comparison to control group C1 (comprising only macrophages), which showed 79±3.6 nanomoles of MDA/mg protein. The mean reduction in MDA levels was highest in probiotic cocktail group T7, comprising all the three probiotic lactobacilli (51%, P <0.01), followed by LCFS cocktail groups (T4-T6) of two lactobacilli (27-40%, P <0.01) and LCFS groups (T1-T3) of individual Lactobacillus (15-27%), respectively (Fig. 2A).
- (A) Estimation of lipid peroxidation (LPO). Outcome on the lipid peroxidation levels (in terms of MDA levels) of rat peritoneal macrophages infected with Shigella dysenteriae type 1 in the presence of lactobacilli cell-free supernatant (LCFS) of probiotic lactobacilli. (B) Estimation of nitrite levels. Effect of LCFS of probiotic lactobacilli on nitrite levels produced in macrophage infected with S. dysenteriae type 1. Values are expressed as mean±standard deviation of three individual values. #P <0.05 compared to negative control group (non infected control group 1, C1), *P <0.01 compared to challenge group (Shigella infected control group 2, C2). MDA, malondialdehyde.
The levels of NO, an indicator of free radical mediated impairment, were determined by estimating the amount of nitrite present in the infected macrophages. As shown in Fig. 2B, decreased levels of nitrite were observed when infected macrophages were treated with LCFS of probiotic lactobacilli. Challenge group C2, composed of macrophage and S. dysenteriae showed 19.47±1.50 μmol of nitrite/mg protein. LCFS cocktail group T7, composed of all the three lactobacilli showed highest and most significant reduction in levels of nitrite (75%, P <0.01), followed by LCFS cocktail groups (T4-T6) of two lactobacilli (67-73%, P <0.01) and LCFS treatment groups (T1-T3) of each lactobacilli (56-63%, P <0.01) taken individually.
Superoxide dismutase (SOD) estimation
The levels of SOD under different treatment groups are shown in Fig. 3A. A significant reduction was observed in levels of SOD in challenge group C2 comprising macrophage and S. dysenteriae (2.06±0.251 units of SOD/mg protein, P <0.05), in comparison to control group C1, comprising only macrophages (4.1±0.2 units of SOD/ mg protein). The levels of SOD were found to be highest and significantly increased (49%, P <0.01) when LCFS cocktail of all the three lactobacilli was employed as a treatment. However, the SOD levels (28-49%, P <0.01 for T6) were relatively low, with treatment groups containing LCFS cocktail of two lactobacilli. No significant increase in levels of SOD was observed on treating infected macrophages with LCFS of L. pentosus and L. paraplantarum, whereas when treated with L. rhamnosus showed a significant increase (48%, P <0.05), respectively.
- (A) Estimation of levels of superoxide dismutase (SOD). Effect of lactobacilli cell-free supernatant (LCFS) of probiotic lactobacilli on SOD levels of macrophage; (B) Estimation of levels of reduced glutathione (GSH): Effect of LCFS of probiotic lactobacilli on GSH levels in macrophages. Values are expressed as mean±standard deviation of three individual values. #P <0.05 compared to negative control group (C1), *P <0.01 compared to challenge group (C2).
Glutathione (GSH) estimation
Levels of GSH in normal macrophages present in control group C1 was found to be 2.133±0.23 μmol GSH/mg protein, whereas when macrophages were infected with S. dysenteriae as in challenge group C2, GSH levels were significantly reduced to 0.5±0.1 μmol GSH/mg protein (P <0.05). As with various treatment measures, the maximum increase in the level of protein GSH was observed with LCFS cocktail of all the three lactobacilli T7 (75%, P <0.01), followed by treatment groups T4-T6 employing LCFS of two lactobacilli (38-72%, P <0.01). The GSH levels in treatment groups of individual Lactobacillus were not significantly increased except with L. rhamnosus treatment (66%, P <0.01) (Fig. 3B).
Discussion
S. dysenteriae is one of the few bacterial pathogens which not only prevents its phagocytosis by macrophages but also effectively survives in them122. The present study indicated that this process could be reversed by treating Shigella-infected macrophages with LCFS of three lactobacilli (L. rhamnosus, L. pentosus and L. paraplantarum) which were isolated from infants' stool samples. The percentage of killing of cells of S. dysenteriae residing in rat peritoneal macrophages varied depending on the type of LCFS used. Maximum killing of S. dysenteriae was observed using LCFS of L. rhamnosus, followed by L. pentosus and L. paraplantarum. However, the killing effect of S. dysenteriae was far more pronounced when cocktail of three lactobacilli was used. The maximum killing of S. dysenteriae was resulted due to the synergistic effect of probiotic LCFS cocktail. The antimicrobial activity of lactobacilli has been attributed to the presence of bacteriocin, lactic acid and H2O2 in the culture filtrates23. The organisms isolated in this study produced lactic acid and H2O2, but not bacteriocin. Our findings were in agreement with others who also showed the prominent role of antimicrobial effect of lactic acid and H2O2 on bacterial pathogens such as Escherichia coli and Salmonella enterica2425. Presence of other molecules such as aldehydes and smaller peptides may also be responsible for antimicrobial activity.
There is substantial evidence that S. dysenteriae infection results in apoptosis of macrophages26 which harbour this bacterial pathogen. The macrophage cell death is attributed to the excessive production of reactive oxygen species and reactive nitrogen intermediates26. In the present study, probiotic treatment of infected macrophages led to a decrease in the level of LPO and nitrite production. L. rhamnosus was most effective to combat free radical-mediated damage in infected macrophages followed by L. pentosus and L. paraplantarum. Although the three lactobacilli reduced levels of LPO and nitrite, the cocktail of three lactobacilli was most effective probably due to their synergistic effect.
The extent of infection depends on the cell's antioxidant status that scavenges the free radicals generated in response to infection27. Probiotics have been reported to enhance antioxidative ability of macrophages infected with bacterial pathogens such as S. typhimurium2829. The potential of probiotics to alter antioxidant status of S. dysenteriae-infected macrophages still remains unexplored. In the current study, lactobacilli isolates restored the levels of SOD and GSH in infected macrophages, hence protecting infected macrophages from free radical-mediated toxicity and apoptosis.
In conclusion, probiotic cocktail of L. pentosus, L. paraplantarum and L. rhamnosus showed anti-Shigella activity against S. dysenteriae type 1 residing inside the rat macrophages. This cocktail has the potential to be used as a natural alternative for treating S. dysenteriae infection, especially in infants for whom stringent antibiotic treatment is not recommended. Further studies need to be done to confirm these finding in vivo.
Acknowledgment
Authors thank Dr R.K. Malik, Microbiology Division, National Dairy Research Institute, Karnal, and Dr Arvind Gulati and Ms Shashi, Institute of Himalayan Bioresource Technology, Palampur, India for guidance and assistance. The first author (RT) acknowledges financial assistance from DST-INSPIRE, India.
Conflicts of Interest: None.
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