Experimental Parasitology 146 (2014) 52–63
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Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r
Full length article
Therapeutic enhancement of newly derived bacteriocins against Giardia lamblia Eglal I. Amer a,*, Shereen F. Mossallam a, Hoda Mahrous b a b
Medical Parasitology Department, Faculty of Medicine, Alexandria University, Egypt Industrial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Sadat City University, Egypt
H I G H L I G H T S
• • • • •
G R A P H I C A L
A B S T R A C T
L. acidophilus bacteriocin showed in vitro activity against G. lamblia trophozoites. Bacteriocin induced severe morphological changes using electron microscopy. Oral bacteriocin for 5 days was sufficient to induce massive parasite reduction. L. acidophilus bacteriocin induced amelioration of intestinal pathology. Bacteriocin holds great promise as an alternative therapy for giardiasis.
A R T I C L E
I N F O
Article history: Received 29 April 2014 Received in revised form 20 September 2014 Accepted 24 September 2014 Available online 6 October 2014 Keywords: Giardia lamblia Bacteriocin Lactobacillus acidophilus Lactobacillus plantarum In vivo In vitro
A B S T R A C T
Trials for identifying efficient anti-giardial agents are still ongoing. Nowadays, bacteriocins have attracted the attention as potential antimicrobial compounds. For the first time, the current study evaluated the therapeutic efficacy of bacteriocins derived from newly isolated Egyptian strains of probiotics Lactobacilli; L. acidophilus (P106) and L. plantarum (P164) against Giardia lamblia. Bacteriocins’ efficacy was evaluated both in vitro; by growth inhibition and adherence assays, and in vivo; through estimation of parasite density, intestinal histopathological examination and ultrastructural analysis of Giardia trophozoites. In vivo bacteriocins’ clinical safety was assessed. In vitro results proved that 50 μg of L. acidophilus bacteriocin induced reduction of the mean Giardia lamblia trophozoites by 58.3 ± 4.04%, while at lower concentrations of 10 and 20 μg of both L. acidophilus and L. plantarum, non significant reduction of the mean parasite density was achieved. In vitro trophozoites adherence was susceptible to the tested bacteriocins at all studied concentrations with variable degrees, while the highest adherence reduction was demonstrated using 50 μg of L acidophilus bacteriocin. In vivo, oral inoculation of 50 μg/mouse L. acidophilus bacteriocin for 5 successive days resulted in a noteworthy decline of the intestinal parasite density, along with amelioration of intestinal pathology of infected mice. Ultrastructural examination proved that
* Corresponding author. Fax: +20 3 4831498. E-mail address: [email protected] (E.I. Amer). http://dx.doi.org/10.1016/j.exppara.2014.09.005 0014-4894/© 2014 Elsevier Inc. All rights reserved.
E.I. Amer et al./Experimental Parasitology 146 (2014) 52–63
53
five doses of L. acidophilus bacteriocin showed marked changes in cellular architecture of the trophozoites with evident disorganization of the cell membrane, adhesive disc and cytoplasmic components. This is the first reported study of the safe anti-giardial efficacy of L. acidophilus (P106) derived bacteriocin, hence highlighting its great promise as a potential therapeutic safe alternative to existing commercial drugs. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Giardiasis, the most common intestinal protozoal infection reported in humans, is considered as one of the important causes of diarrheal disease worldwide. Recently, giardiasis was included in the ‘Neglected Disease Initiative’, estimating that 280 million people are infected each year (Lalle, 2010; Savioli et al., 2006). At present, treatment options include the nitroheterocyclic drugs tinidazole, metronidazole (MNZ) and furazolidone, the substituted acridine, quinacrine, and the benzimidazole, albendazole. Paromomycin is also used in some situations, and nitazoxanide is proving to be useful (Morrone et al., 2004; Sullayman et al., 2002). Among the current recommended treatments, MNZ has been the mainstay of treatment for decades and is still widely used (Escobedo and Cimerman, 2007). However, most of the therapeutically used antigiardial drugs, including MNZ, cause severe side effects and are not well tolerated by many patients. Furthermore, existing antimicrobial therapies are not always effective, and drug resistance to all available drugs has been demonstrated in the laboratory (Petri, 2003). In addition, clinical resistance has been reported, including cases where patients failed both MNZ and albendazole treatments (Tian et al., 2010). Hence, identifying new anti-giardial agents is an important consideration for the future (Tejman-Yarden and Eckmann, 2011). Some bacteria, fungi, plants, insects and vertebrates excrete antimicrobial peptides, which are known to have inhibitory effects directed against enveloped viruses, bacteria, fungi, parasites as well as cancer cells (Pálffy et al., 2009). Bacteriocins are a diverse family of ribosomally synthesized proteins produced by probiotic bacteria. According to the generally accepted definition, probiotics are live microorganisms beneficially affecting the health of a host animal (Fuller, 1989). To be effective, probiotic bacteria must exhibit a number of functional characteristics, including the resistance to technologic processes, gastric acidity, bile toxicity and the ability to adhere to the intestinal epithelium. Several reports have indicated that the survival and viability of bacteria from most probiotic products in gastric juice is rather poor, and only few of them exhibit stability and resistance to adverse environmental conditions (Dunne et al., 2001; Nighswogner et al., 1996). Therefore, bacteriocins derived from probiotic bacteria could provide the beneficial role of bacteria away from these conditions. Although research in this area is still in its infancy, there is intriguing evidence to suggest that bacteriocins are characterized by their antimicrobial activity, most frequently against bacteria which are phylogenetically closely related (narrow spectrum of activity). However, a surprisingly high level of inter-specific killing was observed; with almost half of the bacteriocins affect more than one taxon. Furthermore, the relationship between killing ability and phylogenetic distance is nonlinear. This nonlinear relationship indicates killing outside of the producer strain’s own species. The observation of a broad killing range for numerous enteric bacteriocins requires that the ecological role proposed for bacteriocins should be reconsidered (Dawson and Scott, 2012; Dobson et al., 2012). Nowadays, bacteriocins have attracted the attention as potential substitutes for, or as combined therapy to currently used antimicrobial compounds due to their powerful killing activity, stability and low toxicity to humans (Hassan et al., 2012; Joerger, 2003). Therefore, this study was designed to evaluate, for the first time, the possible in vitro and in vivo therapeutic efficacy of bacteriocins derived from newly isolated Egyptian strains
of probiotics L. acidophilus (P106) and L. plantarum (P164) against Giardia lamblia (G. lamblia). 2. Material and methods 2.1. Bacteriocins 2.1.1. Probiotic strains isolation and maintenance Two probiotic strains; L. acidophilus and L. plantarum have been isolated from healthy breast-fed 15 day old Egyptian infants, and were identified as P106 and P164; respectively by Mahrous (2006). They were used after the selection had been done according to Bergey’s Manual of Determinative Bacteriology, 9th edition with confirmation of the identification by SDS-PAGE technique and API System (Holt et al., 1994). The strains were tested for their probiotic characteristic i.e. gastric acid resistance, bile salt tolerance, antibacterial activity, adhesion to human mucus. Their antimicrobial activity was determined by measuring the diameter of the inhibition zone around the wells of Gram negative E. coli ATCC25922 and Gram positive Bacillus subtilis NIB3610. Lactobacillus strains were cultivated in MRS (de Man Rogosa Sharpe) broth (Lab M, IDG, UK) and incubated at 37 °C in BBL anaerobic jar (Becton Dickinson Microbiology Systems, Sparks, MD) provided with disposable BBL gas generating pack (CO2 system envelopes, Oxoid, Ltd., West Heidelberg, VIC, Canada). Isolates were stored at −20 °C in MRS broth supplemented with 25% (v/v) glycerol. For routine analysis, the strains were subcultured twice in MRS broth at 37 °C for 24 h. 2.1.2. Bacteriocin extraction, preparation and purification Isolated L. acidophilus and L. plantarum strains were grown in MRS broth at 37 °C for 24 h. After incubation, the bacterial cells were removed by centrifugation and 200 mL of the crude bacteriocins supernatant was treated with 1.4 g streptomycin sulfate for nucleic acid precipitation. The supernatant was fractionated by the addition of 525 g solid ammonium sulfate with continuous stirring to get 80% saturation. The precipitated fraction was collected by centrifugation at 10,000 rpm for 10 min at 4 °C and dialyzed against 10 volumes of phosphate buffer pH 7.5. The buffer was changed several times, until equilibration occurred after overnight dialysis. The dialyzed solution was used to find the concentration of protein. Further purification was carried out using column chromatography with a Superdex-G 200 packed column (1.5 × 3 cm), using 0.1 M phosphate buffer (pH = 7) as the eluant. The purified sample was dissolved in the eluting buffer at 10 mL and applied to the gel permeation column at room temperature. An elution flow rate of 1.5 mL/ 15 min was employed and fractions were collected sequentially, and monitored by UV absorption at 280 nm. The bacteriocin-containing samples (fractions 15–25) were pooled and the resultant solutions were dialyzed against distilled water (molecular weight cutoff 10 kDa) overnight (Van Reenen et al., 1998). 2.1.3. Bacteriocin activity units Twofold serial dilutions of cell free neutralized supernatant of the producer strain and of partially purified (80% ammonium sulfate precipitated) bacteriocins were prepared and were followed for agar well diffusion assay. Plates were incubated overnight at 30 °C and zones of inhibition around each well were measured in mm. The bacteriocin titer was expressed as arbitrary or activity unit/mL. One arbitrary unit (AU) of bacteriocin is defined as the reciprocal of the
54
E.I. Amer et al./Experimental Parasitology 146 (2014) 52–63
last serial dilution demonstrating significant inhibitory activity (Barefoot and Klaenhammer, 1983).
AU ml =
Reciprocal of the highest dilution × 1000 Volume of bacteriocin added
2.2. Parasites purification and maintenance 2.2.1. Giardia lamblia cysts They were obtained from a patient attending the Tropical Medicine Department, Alexandria Main University Hospital, Egypt, suffering chronic diarrhea. Cysts were concentrated and purified using sucrose flotation method. The collected cysts were washed twice in distilled water and sedimented by centrifugation at 800 g for 10 min. After the supernatant was discarded, the pellet was resuspended in a sterile tube containing 3% sodium hypochlorite to kill other microorganisms. Tubes containing Giardia cysts in 3% sodium hypochlorite solution were chilled in an ice bath for 10 min and then were washed three times with a phosphate buffer saline that was free of calcium and magnesium. The process of purification was performed under aseptic conditions (Buraud et al., 1991). 2.2.2. Giardia lamblia trophozoites They were obtained by oral inoculation of two laboratory bred Swiss albino mice 4–6 weeks old with 2 × 105 G. lamblia cysts. Six days post infection, mice were sacrificed and the entire small intestine was removed, longitudinally opened and placed in 10 mL of cold saline for at least 2 h. Tubes were vortexed to ensure complete detachment of the parasites (Leméea et al., 2000). Giardia trophozoites were separated from the mucosa by gauze filtration and filtrates were routinely cultured on TYI-S-33 medium supplemented with bile salts, 10% heat inactivated bovine serum, 10 μg of ampicillin per mL and 10 μg of gentamicin sulfate per mL, under anaerobic conditions at 37 °C (Cedillo et al., 1991; Keister, 1983). After 2 days, trophozoites were harvested from the cultures by cooling the culture vials in an iced water bath for 5 min, followed by centrifugation at 1500 rpm for 10 min. Trophozoites were washed three times in Hanks’ balanced salt solution (HBSS, pH 7.1) and their count was performed in a hemocytometer (Neubaeur cell-counter chamber). These trophozoites were used as inoculums for both in vitro and in vivo assays.
Whereas, C1 represents the concentration of Giardia lamblia trophozoites in treated culture and C0 represents the concentration of the parasites in the initial inoculums. 2.4.2. Adherence assay The direct effect of bacteriocins at different studied concentrations on adherence of G. lamblia trophozoites was evaluated as previously described by Khademi et al., 2006, in comparison with MNZ. Inoculums of 104/mL trophozoites were transferred into culture tubes. The tested concentrations of each bacteriocin were pipetted into these tubes and were incubated at 37 °C, for 2 h. The effluents were collected in disposable test tubes, adherent cells were dislodged by 10-min incubation in ice-cold HBSS, and numbers of attached cells was microscopically determined using a hemocytometer. The experiments were performed three times and the mean results calculated as follows:
Percentage adherence = 100 −
Ce × 100 Co
Where, Ce represents the concentration of Giardia in the effluent, and Co represents the concentration in the original suspension. 2.5. In vivo assay 2.5.1. Experimental animals The in vivo anti-giardial activity of the extracted bacteriocins was tested using 120 laboratory bred Swiss strain albino mice of both sexes, 4–6 weeks old and weighing 20–25 g. These mice were obtained from the animal house of the Medical Parasitology Department, Faculty of Medicine, Alexandria University, Egypt. Animals were kept under standard conditions. The experimental protocol was approved by the ethics committee of Faculty of Medicine, Alexandria University, Egypt. Animal experiment complied with the Egyptian national regulations in accordance with the guidelines for care and use of laboratory animals. Prior to the experimental infection, mice were orally treated for 3 consecutive days with mebendazole solution (10 mg/mouse/day). One week after treatment, three consecutive fecal examinations were performed to ensure that mice were free from all possible protozoal infections. Experimental mice were equally divided into four groups; 2.5.1.1. Group I. Thirty infected non-treated control mice.
2.3. MNZ (Flagyl®) intravenous infusion sterile solution 500 mg/mL, Amriya for pharmaceutical industries, Alexandria, Egypt, was purchased from a local pharmacy.
2.5.1.2. Group II. Thirty infected MNZ-treated therapeutic control mice. 2.5.1.3. Group III. Thirty infected L. acidophilus-bacteriocin treated experimental mice.
2.4. In vitro assay 2.4.1. Growth inhibition assay For susceptibility tests, trophozoites from a logarithmically growing culture were adjusted as 104 per milliliter and transferred to fresh medium. Final dilutions of bacteriocins and MNZ were freshly prepared in PBS immediately before being incubated with parasites at different concentrations of 10, 20 and 50 μg/mL at 37 °C for 24 h, after which they were chilled and the trophozoites concentration was determined using an improved double Neubauer ruling hemocytometer. Experiments were performed in triplicates. For each test, a control series was included in the experiment and the results were calculated as follows (Khademi et al., 2006)
Percentage growth inhibition = 100 −
C1 × 100 C0
2.5.1.4. Group IV. Thirty infected L. plantarum-bacteriocin treated experimental mice. All mice were orally inoculated with single dose of 2 × 105/ 0.1 mL G. lamblia trophozoites (Leméea et al., 2000). On the 6th day post infection, infected treated groups were orally inoculated with either 50 mg/kg/0.1 mL MNZ (GII), or 50 μg/0.1 mL/mouse bacteriocins (GIII and IV). These infected treated mice (GII, III and IV) were further subdivided into three subgroups (10 mice each) according to the duration of therapy; 1 day therapy (subgroup a), 3 successive days therapy (subgroup b), and 5 successive days therapy (subgroup c). All treated mice were sacrificed 48 h after the last therapeutic dose. Simultaneously, 10 mice of the infected non-treated control group (GI) were sacrificed at each of the following durations; 9th, 11th, 13th post infection; corresponding to the treated subgroups. The experiment was repeated thrice. Data of one of the
E.I. Amer et al./Experimental Parasitology 146 (2014) 52–63
three independent experiments, having the average replicate, were presented. The entire small intestine of mice belonging to each group was removed, a small part of which was fixed in 10% neutral buffered formalin and paraffin embedded 5 μm sections were serially cut and stained with hematoxylin and eosin. Grading of histopathological changes in intestinal mucosa was performed according to Oberhuber and Stolte (1990) and Handousa et al. (2003). Briefly, Grade 0: normal architecture of villi, with villous/crypt ratio 3:1. Grade I: mostly finger shaped villi having focal villous flattening, and/or broadening, with mild lymphocytic infiltrate. Epithelium of some villi showed nuclear shift from basal to superficial part of the cell. Grade II: mostly, short broad or club shaped villi, with moderate inflammatory infiltrate and intraepithelial lymphocytes. Grade III: fused or bow shaped villi, with a moderate to severe inflammatory infiltrate in lamina propria. Grade IV: villous atrophy in areas and sloughing of brush border in others, i.e. no definite villi in flattened mucosa, except for some indentations and crypts opening on the surface with occasional crypt abscess formation and dense infiltrate in lamina propria. Giardia trophozoites were separated from the mucosa of the remaining intestinal part as previously mentioned (Leméea et al., 2000) and the mean parasite density and in vivo reduction percentage were estimated for of all groups. Trophozoite pellets, collected from mice treated with L. acidophilus bacteriocin for 5 successive days, were fixed in cold 1.5% glutaraldehyde and were kept at 4 °C overnight, then were washed three times in distilled water and contrasted in saturated uranyl acetate for 30 min for Joel TEM, or directly dehydrated in an ethanol series (50%, 70%, 90%, 3X 100%) for SEM (Hayat, 1981).
55
tion, food intake, weight gain and general health status (hair loss and behavioral changes) of these animals were daily monitored. One day post treatment, mice were sacrificed to perform gastric and intestinal histological analysis; in order to detect any possible evidence of pathological changes (Maragkoudakis et al., 2009). 2.7. Statistical analysis Computer program SPSS for windows, release 18.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis. All numeric variables were expressed as mean ± standard deviation (SD). Comparison of different variables in various groups was done using Mann–Whitney test for nonparametric variables. 3. Results 3.1. In vitro effectiveness
2.6. In vivo assessment of bacteriocins’ clinical safety
3.1.1. Growth inhibition assay Bacteriocins’ susceptibility was investigated in cultured G. lamblia trophozoites to determine the percentage reduction of the mean parasite density after 24 h of incubation. Under the same conditions, untreated control culture tubes showed 15.33 ± 3.8% parasites growth. At low concentrations of 10 and 20 μg, bacteriocins isolated from both tested strains succeeded to inhibit further growth of the parasites without significant reduction of their numbers. Whereas, at 50 μg bacteriocin derived from L. acidophilus induced growth inhibition of 58.3 ± 4.04%; in comparison with those achieved by bacteriocin isolated from L. plantarum (29.06 ± 3.5%). MNZ was superior of all, as it induced complete lysis of the parasites under the same concentrations (Fig. 1).
Prior to the in vivo susceptibility assay, bacteriocins’ safety measures were performed. Thirty naive non-infected mice were equally divided into three subgroups for assessing in vivo safety of maximal chosen duration of the tested bacteriocins. Mice were fed with 0.1 mL/mouse sterile broth, 50 μg/0.1 mL/mouse L. acidophilus, L. plantarum bacteriocins; respectively, for 5 days. Water consump-
3.1.2. Adherence assay In vitro adherence of G. lamblia trophozoites to the control culture tubes after 2 h incubation was 46.67%. Bacteriocins isolated from both L. acidophilus and L. plantarum showed ability to induce adherence reduction, irrespective of their ability to affect trophozoite growth inhibition. A direct relationship was shown between the
Fig. 1. Percentage reduction of the in vitro assay induced by the different therapeutic agents against Giardia lamblia trophozoites.
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E.I. Amer et al./Experimental Parasitology 146 (2014) 52–63
tested concentrations and the adherence reduction assay. At high concentration of 50 μg, the adherence percentages were 17.33 ± 3.7 and 29.67 ± 0.58 respectively (Table 1). 3.2. In vivo effectiveness Daily observation of the food intake and clinical behavior of noninfected mice; which received either strain of bacteriocins, revealed no changes in the water consumption, food intake, weight gain or their general behavior and well-being; as compared with those receiving sterile broth. Histologic analysis detected neither gastric nor intestinal inflammation in these animals. Giardial in vivo growth inhibition was assessed by recording the mean count of G. lamblia trophozoites collected from the intestinal mucosa of the studied groups. In comparison with the infected untreated control, the results showed that bacteriocin isolated from L. acidophilus induced reduction of G. lamblia trophozoites growth with variable degrees. Its efficacy was dependent on the duration of therapy; the highest reduction percentage was achieved 48 h after the five dosesregimens (81.63%). On the other hand, bacteriocin isolated from L. plantarum failed to induce significant reduction of the parasite count at both single and triple doses regimens, while its efficacy at five doses was not comparable with that of bacteriocin isolated from L. acidophilus under the same circumstances (31.38%). In comparison with
Table 1 The adherence percentage among different studied therapies at different concentration. Concentrations
10 μg Min.–Max. Mean ± SD Median 20 μg Min.–Max. Mean ± SD Median 50 μg Min.–Max. Mean ± SD Median
% Adherence
p
L. acidophilus bacteriocin
L. plantarum bacteriocin
MNZ
25.0–35.0 30.0 ± 5.0 30.0
39.0–44.0 41.0a ± 2.65 40.0
23.0–30.0 27.0b ± 3.61 28.0
0.010*
20.0–29.0 23.67 ± 4.73 22.0
34.0–39.0 36.67a ± 2.52 37.0
15.0–20.0 18.0b ± 2.65 19.0
0.002*
13.0–20.0 17.33 ± 3.79 19.0
29.0–30.0 29.67a ± 0.58 30.0
10.0–12.0 11.33a,b ± 1.15 12.0
Contents lists available at ScienceDirect
Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r
Full length article
Therapeutic enhancement of newly derived bacteriocins against Giardia lamblia Eglal I. Amer a,*, Shereen F. Mossallam a, Hoda Mahrous b a b
Medical Parasitology Department, Faculty of Medicine, Alexandria University, Egypt Industrial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Sadat City University, Egypt
H I G H L I G H T S
• • • • •
G R A P H I C A L
A B S T R A C T
L. acidophilus bacteriocin showed in vitro activity against G. lamblia trophozoites. Bacteriocin induced severe morphological changes using electron microscopy. Oral bacteriocin for 5 days was sufficient to induce massive parasite reduction. L. acidophilus bacteriocin induced amelioration of intestinal pathology. Bacteriocin holds great promise as an alternative therapy for giardiasis.
A R T I C L E
I N F O
Article history: Received 29 April 2014 Received in revised form 20 September 2014 Accepted 24 September 2014 Available online 6 October 2014 Keywords: Giardia lamblia Bacteriocin Lactobacillus acidophilus Lactobacillus plantarum In vivo In vitro
A B S T R A C T
Trials for identifying efficient anti-giardial agents are still ongoing. Nowadays, bacteriocins have attracted the attention as potential antimicrobial compounds. For the first time, the current study evaluated the therapeutic efficacy of bacteriocins derived from newly isolated Egyptian strains of probiotics Lactobacilli; L. acidophilus (P106) and L. plantarum (P164) against Giardia lamblia. Bacteriocins’ efficacy was evaluated both in vitro; by growth inhibition and adherence assays, and in vivo; through estimation of parasite density, intestinal histopathological examination and ultrastructural analysis of Giardia trophozoites. In vivo bacteriocins’ clinical safety was assessed. In vitro results proved that 50 μg of L. acidophilus bacteriocin induced reduction of the mean Giardia lamblia trophozoites by 58.3 ± 4.04%, while at lower concentrations of 10 and 20 μg of both L. acidophilus and L. plantarum, non significant reduction of the mean parasite density was achieved. In vitro trophozoites adherence was susceptible to the tested bacteriocins at all studied concentrations with variable degrees, while the highest adherence reduction was demonstrated using 50 μg of L acidophilus bacteriocin. In vivo, oral inoculation of 50 μg/mouse L. acidophilus bacteriocin for 5 successive days resulted in a noteworthy decline of the intestinal parasite density, along with amelioration of intestinal pathology of infected mice. Ultrastructural examination proved that
* Corresponding author. Fax: +20 3 4831498. E-mail address: [email protected] (E.I. Amer). http://dx.doi.org/10.1016/j.exppara.2014.09.005 0014-4894/© 2014 Elsevier Inc. All rights reserved.
E.I. Amer et al./Experimental Parasitology 146 (2014) 52–63
53
five doses of L. acidophilus bacteriocin showed marked changes in cellular architecture of the trophozoites with evident disorganization of the cell membrane, adhesive disc and cytoplasmic components. This is the first reported study of the safe anti-giardial efficacy of L. acidophilus (P106) derived bacteriocin, hence highlighting its great promise as a potential therapeutic safe alternative to existing commercial drugs. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Giardiasis, the most common intestinal protozoal infection reported in humans, is considered as one of the important causes of diarrheal disease worldwide. Recently, giardiasis was included in the ‘Neglected Disease Initiative’, estimating that 280 million people are infected each year (Lalle, 2010; Savioli et al., 2006). At present, treatment options include the nitroheterocyclic drugs tinidazole, metronidazole (MNZ) and furazolidone, the substituted acridine, quinacrine, and the benzimidazole, albendazole. Paromomycin is also used in some situations, and nitazoxanide is proving to be useful (Morrone et al., 2004; Sullayman et al., 2002). Among the current recommended treatments, MNZ has been the mainstay of treatment for decades and is still widely used (Escobedo and Cimerman, 2007). However, most of the therapeutically used antigiardial drugs, including MNZ, cause severe side effects and are not well tolerated by many patients. Furthermore, existing antimicrobial therapies are not always effective, and drug resistance to all available drugs has been demonstrated in the laboratory (Petri, 2003). In addition, clinical resistance has been reported, including cases where patients failed both MNZ and albendazole treatments (Tian et al., 2010). Hence, identifying new anti-giardial agents is an important consideration for the future (Tejman-Yarden and Eckmann, 2011). Some bacteria, fungi, plants, insects and vertebrates excrete antimicrobial peptides, which are known to have inhibitory effects directed against enveloped viruses, bacteria, fungi, parasites as well as cancer cells (Pálffy et al., 2009). Bacteriocins are a diverse family of ribosomally synthesized proteins produced by probiotic bacteria. According to the generally accepted definition, probiotics are live microorganisms beneficially affecting the health of a host animal (Fuller, 1989). To be effective, probiotic bacteria must exhibit a number of functional characteristics, including the resistance to technologic processes, gastric acidity, bile toxicity and the ability to adhere to the intestinal epithelium. Several reports have indicated that the survival and viability of bacteria from most probiotic products in gastric juice is rather poor, and only few of them exhibit stability and resistance to adverse environmental conditions (Dunne et al., 2001; Nighswogner et al., 1996). Therefore, bacteriocins derived from probiotic bacteria could provide the beneficial role of bacteria away from these conditions. Although research in this area is still in its infancy, there is intriguing evidence to suggest that bacteriocins are characterized by their antimicrobial activity, most frequently against bacteria which are phylogenetically closely related (narrow spectrum of activity). However, a surprisingly high level of inter-specific killing was observed; with almost half of the bacteriocins affect more than one taxon. Furthermore, the relationship between killing ability and phylogenetic distance is nonlinear. This nonlinear relationship indicates killing outside of the producer strain’s own species. The observation of a broad killing range for numerous enteric bacteriocins requires that the ecological role proposed for bacteriocins should be reconsidered (Dawson and Scott, 2012; Dobson et al., 2012). Nowadays, bacteriocins have attracted the attention as potential substitutes for, or as combined therapy to currently used antimicrobial compounds due to their powerful killing activity, stability and low toxicity to humans (Hassan et al., 2012; Joerger, 2003). Therefore, this study was designed to evaluate, for the first time, the possible in vitro and in vivo therapeutic efficacy of bacteriocins derived from newly isolated Egyptian strains
of probiotics L. acidophilus (P106) and L. plantarum (P164) against Giardia lamblia (G. lamblia). 2. Material and methods 2.1. Bacteriocins 2.1.1. Probiotic strains isolation and maintenance Two probiotic strains; L. acidophilus and L. plantarum have been isolated from healthy breast-fed 15 day old Egyptian infants, and were identified as P106 and P164; respectively by Mahrous (2006). They were used after the selection had been done according to Bergey’s Manual of Determinative Bacteriology, 9th edition with confirmation of the identification by SDS-PAGE technique and API System (Holt et al., 1994). The strains were tested for their probiotic characteristic i.e. gastric acid resistance, bile salt tolerance, antibacterial activity, adhesion to human mucus. Their antimicrobial activity was determined by measuring the diameter of the inhibition zone around the wells of Gram negative E. coli ATCC25922 and Gram positive Bacillus subtilis NIB3610. Lactobacillus strains were cultivated in MRS (de Man Rogosa Sharpe) broth (Lab M, IDG, UK) and incubated at 37 °C in BBL anaerobic jar (Becton Dickinson Microbiology Systems, Sparks, MD) provided with disposable BBL gas generating pack (CO2 system envelopes, Oxoid, Ltd., West Heidelberg, VIC, Canada). Isolates were stored at −20 °C in MRS broth supplemented with 25% (v/v) glycerol. For routine analysis, the strains were subcultured twice in MRS broth at 37 °C for 24 h. 2.1.2. Bacteriocin extraction, preparation and purification Isolated L. acidophilus and L. plantarum strains were grown in MRS broth at 37 °C for 24 h. After incubation, the bacterial cells were removed by centrifugation and 200 mL of the crude bacteriocins supernatant was treated with 1.4 g streptomycin sulfate for nucleic acid precipitation. The supernatant was fractionated by the addition of 525 g solid ammonium sulfate with continuous stirring to get 80% saturation. The precipitated fraction was collected by centrifugation at 10,000 rpm for 10 min at 4 °C and dialyzed against 10 volumes of phosphate buffer pH 7.5. The buffer was changed several times, until equilibration occurred after overnight dialysis. The dialyzed solution was used to find the concentration of protein. Further purification was carried out using column chromatography with a Superdex-G 200 packed column (1.5 × 3 cm), using 0.1 M phosphate buffer (pH = 7) as the eluant. The purified sample was dissolved in the eluting buffer at 10 mL and applied to the gel permeation column at room temperature. An elution flow rate of 1.5 mL/ 15 min was employed and fractions were collected sequentially, and monitored by UV absorption at 280 nm. The bacteriocin-containing samples (fractions 15–25) were pooled and the resultant solutions were dialyzed against distilled water (molecular weight cutoff 10 kDa) overnight (Van Reenen et al., 1998). 2.1.3. Bacteriocin activity units Twofold serial dilutions of cell free neutralized supernatant of the producer strain and of partially purified (80% ammonium sulfate precipitated) bacteriocins were prepared and were followed for agar well diffusion assay. Plates were incubated overnight at 30 °C and zones of inhibition around each well were measured in mm. The bacteriocin titer was expressed as arbitrary or activity unit/mL. One arbitrary unit (AU) of bacteriocin is defined as the reciprocal of the
54
E.I. Amer et al./Experimental Parasitology 146 (2014) 52–63
last serial dilution demonstrating significant inhibitory activity (Barefoot and Klaenhammer, 1983).
AU ml =
Reciprocal of the highest dilution × 1000 Volume of bacteriocin added
2.2. Parasites purification and maintenance 2.2.1. Giardia lamblia cysts They were obtained from a patient attending the Tropical Medicine Department, Alexandria Main University Hospital, Egypt, suffering chronic diarrhea. Cysts were concentrated and purified using sucrose flotation method. The collected cysts were washed twice in distilled water and sedimented by centrifugation at 800 g for 10 min. After the supernatant was discarded, the pellet was resuspended in a sterile tube containing 3% sodium hypochlorite to kill other microorganisms. Tubes containing Giardia cysts in 3% sodium hypochlorite solution were chilled in an ice bath for 10 min and then were washed three times with a phosphate buffer saline that was free of calcium and magnesium. The process of purification was performed under aseptic conditions (Buraud et al., 1991). 2.2.2. Giardia lamblia trophozoites They were obtained by oral inoculation of two laboratory bred Swiss albino mice 4–6 weeks old with 2 × 105 G. lamblia cysts. Six days post infection, mice were sacrificed and the entire small intestine was removed, longitudinally opened and placed in 10 mL of cold saline for at least 2 h. Tubes were vortexed to ensure complete detachment of the parasites (Leméea et al., 2000). Giardia trophozoites were separated from the mucosa by gauze filtration and filtrates were routinely cultured on TYI-S-33 medium supplemented with bile salts, 10% heat inactivated bovine serum, 10 μg of ampicillin per mL and 10 μg of gentamicin sulfate per mL, under anaerobic conditions at 37 °C (Cedillo et al., 1991; Keister, 1983). After 2 days, trophozoites were harvested from the cultures by cooling the culture vials in an iced water bath for 5 min, followed by centrifugation at 1500 rpm for 10 min. Trophozoites were washed three times in Hanks’ balanced salt solution (HBSS, pH 7.1) and their count was performed in a hemocytometer (Neubaeur cell-counter chamber). These trophozoites were used as inoculums for both in vitro and in vivo assays.
Whereas, C1 represents the concentration of Giardia lamblia trophozoites in treated culture and C0 represents the concentration of the parasites in the initial inoculums. 2.4.2. Adherence assay The direct effect of bacteriocins at different studied concentrations on adherence of G. lamblia trophozoites was evaluated as previously described by Khademi et al., 2006, in comparison with MNZ. Inoculums of 104/mL trophozoites were transferred into culture tubes. The tested concentrations of each bacteriocin were pipetted into these tubes and were incubated at 37 °C, for 2 h. The effluents were collected in disposable test tubes, adherent cells were dislodged by 10-min incubation in ice-cold HBSS, and numbers of attached cells was microscopically determined using a hemocytometer. The experiments were performed three times and the mean results calculated as follows:
Percentage adherence = 100 −
Ce × 100 Co
Where, Ce represents the concentration of Giardia in the effluent, and Co represents the concentration in the original suspension. 2.5. In vivo assay 2.5.1. Experimental animals The in vivo anti-giardial activity of the extracted bacteriocins was tested using 120 laboratory bred Swiss strain albino mice of both sexes, 4–6 weeks old and weighing 20–25 g. These mice were obtained from the animal house of the Medical Parasitology Department, Faculty of Medicine, Alexandria University, Egypt. Animals were kept under standard conditions. The experimental protocol was approved by the ethics committee of Faculty of Medicine, Alexandria University, Egypt. Animal experiment complied with the Egyptian national regulations in accordance with the guidelines for care and use of laboratory animals. Prior to the experimental infection, mice were orally treated for 3 consecutive days with mebendazole solution (10 mg/mouse/day). One week after treatment, three consecutive fecal examinations were performed to ensure that mice were free from all possible protozoal infections. Experimental mice were equally divided into four groups; 2.5.1.1. Group I. Thirty infected non-treated control mice.
2.3. MNZ (Flagyl®) intravenous infusion sterile solution 500 mg/mL, Amriya for pharmaceutical industries, Alexandria, Egypt, was purchased from a local pharmacy.
2.5.1.2. Group II. Thirty infected MNZ-treated therapeutic control mice. 2.5.1.3. Group III. Thirty infected L. acidophilus-bacteriocin treated experimental mice.
2.4. In vitro assay 2.4.1. Growth inhibition assay For susceptibility tests, trophozoites from a logarithmically growing culture were adjusted as 104 per milliliter and transferred to fresh medium. Final dilutions of bacteriocins and MNZ were freshly prepared in PBS immediately before being incubated with parasites at different concentrations of 10, 20 and 50 μg/mL at 37 °C for 24 h, after which they were chilled and the trophozoites concentration was determined using an improved double Neubauer ruling hemocytometer. Experiments were performed in triplicates. For each test, a control series was included in the experiment and the results were calculated as follows (Khademi et al., 2006)
Percentage growth inhibition = 100 −
C1 × 100 C0
2.5.1.4. Group IV. Thirty infected L. plantarum-bacteriocin treated experimental mice. All mice were orally inoculated with single dose of 2 × 105/ 0.1 mL G. lamblia trophozoites (Leméea et al., 2000). On the 6th day post infection, infected treated groups were orally inoculated with either 50 mg/kg/0.1 mL MNZ (GII), or 50 μg/0.1 mL/mouse bacteriocins (GIII and IV). These infected treated mice (GII, III and IV) were further subdivided into three subgroups (10 mice each) according to the duration of therapy; 1 day therapy (subgroup a), 3 successive days therapy (subgroup b), and 5 successive days therapy (subgroup c). All treated mice were sacrificed 48 h after the last therapeutic dose. Simultaneously, 10 mice of the infected non-treated control group (GI) were sacrificed at each of the following durations; 9th, 11th, 13th post infection; corresponding to the treated subgroups. The experiment was repeated thrice. Data of one of the
E.I. Amer et al./Experimental Parasitology 146 (2014) 52–63
three independent experiments, having the average replicate, were presented. The entire small intestine of mice belonging to each group was removed, a small part of which was fixed in 10% neutral buffered formalin and paraffin embedded 5 μm sections were serially cut and stained with hematoxylin and eosin. Grading of histopathological changes in intestinal mucosa was performed according to Oberhuber and Stolte (1990) and Handousa et al. (2003). Briefly, Grade 0: normal architecture of villi, with villous/crypt ratio 3:1. Grade I: mostly finger shaped villi having focal villous flattening, and/or broadening, with mild lymphocytic infiltrate. Epithelium of some villi showed nuclear shift from basal to superficial part of the cell. Grade II: mostly, short broad or club shaped villi, with moderate inflammatory infiltrate and intraepithelial lymphocytes. Grade III: fused or bow shaped villi, with a moderate to severe inflammatory infiltrate in lamina propria. Grade IV: villous atrophy in areas and sloughing of brush border in others, i.e. no definite villi in flattened mucosa, except for some indentations and crypts opening on the surface with occasional crypt abscess formation and dense infiltrate in lamina propria. Giardia trophozoites were separated from the mucosa of the remaining intestinal part as previously mentioned (Leméea et al., 2000) and the mean parasite density and in vivo reduction percentage were estimated for of all groups. Trophozoite pellets, collected from mice treated with L. acidophilus bacteriocin for 5 successive days, were fixed in cold 1.5% glutaraldehyde and were kept at 4 °C overnight, then were washed three times in distilled water and contrasted in saturated uranyl acetate for 30 min for Joel TEM, or directly dehydrated in an ethanol series (50%, 70%, 90%, 3X 100%) for SEM (Hayat, 1981).
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tion, food intake, weight gain and general health status (hair loss and behavioral changes) of these animals were daily monitored. One day post treatment, mice were sacrificed to perform gastric and intestinal histological analysis; in order to detect any possible evidence of pathological changes (Maragkoudakis et al., 2009). 2.7. Statistical analysis Computer program SPSS for windows, release 18.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis. All numeric variables were expressed as mean ± standard deviation (SD). Comparison of different variables in various groups was done using Mann–Whitney test for nonparametric variables. 3. Results 3.1. In vitro effectiveness
2.6. In vivo assessment of bacteriocins’ clinical safety
3.1.1. Growth inhibition assay Bacteriocins’ susceptibility was investigated in cultured G. lamblia trophozoites to determine the percentage reduction of the mean parasite density after 24 h of incubation. Under the same conditions, untreated control culture tubes showed 15.33 ± 3.8% parasites growth. At low concentrations of 10 and 20 μg, bacteriocins isolated from both tested strains succeeded to inhibit further growth of the parasites without significant reduction of their numbers. Whereas, at 50 μg bacteriocin derived from L. acidophilus induced growth inhibition of 58.3 ± 4.04%; in comparison with those achieved by bacteriocin isolated from L. plantarum (29.06 ± 3.5%). MNZ was superior of all, as it induced complete lysis of the parasites under the same concentrations (Fig. 1).
Prior to the in vivo susceptibility assay, bacteriocins’ safety measures were performed. Thirty naive non-infected mice were equally divided into three subgroups for assessing in vivo safety of maximal chosen duration of the tested bacteriocins. Mice were fed with 0.1 mL/mouse sterile broth, 50 μg/0.1 mL/mouse L. acidophilus, L. plantarum bacteriocins; respectively, for 5 days. Water consump-
3.1.2. Adherence assay In vitro adherence of G. lamblia trophozoites to the control culture tubes after 2 h incubation was 46.67%. Bacteriocins isolated from both L. acidophilus and L. plantarum showed ability to induce adherence reduction, irrespective of their ability to affect trophozoite growth inhibition. A direct relationship was shown between the
Fig. 1. Percentage reduction of the in vitro assay induced by the different therapeutic agents against Giardia lamblia trophozoites.
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E.I. Amer et al./Experimental Parasitology 146 (2014) 52–63
tested concentrations and the adherence reduction assay. At high concentration of 50 μg, the adherence percentages were 17.33 ± 3.7 and 29.67 ± 0.58 respectively (Table 1). 3.2. In vivo effectiveness Daily observation of the food intake and clinical behavior of noninfected mice; which received either strain of bacteriocins, revealed no changes in the water consumption, food intake, weight gain or their general behavior and well-being; as compared with those receiving sterile broth. Histologic analysis detected neither gastric nor intestinal inflammation in these animals. Giardial in vivo growth inhibition was assessed by recording the mean count of G. lamblia trophozoites collected from the intestinal mucosa of the studied groups. In comparison with the infected untreated control, the results showed that bacteriocin isolated from L. acidophilus induced reduction of G. lamblia trophozoites growth with variable degrees. Its efficacy was dependent on the duration of therapy; the highest reduction percentage was achieved 48 h after the five dosesregimens (81.63%). On the other hand, bacteriocin isolated from L. plantarum failed to induce significant reduction of the parasite count at both single and triple doses regimens, while its efficacy at five doses was not comparable with that of bacteriocin isolated from L. acidophilus under the same circumstances (31.38%). In comparison with
Table 1 The adherence percentage among different studied therapies at different concentration. Concentrations
10 μg Min.–Max. Mean ± SD Median 20 μg Min.–Max. Mean ± SD Median 50 μg Min.–Max. Mean ± SD Median
% Adherence
p
L. acidophilus bacteriocin
L. plantarum bacteriocin
MNZ
25.0–35.0 30.0 ± 5.0 30.0
39.0–44.0 41.0a ± 2.65 40.0
23.0–30.0 27.0b ± 3.61 28.0
0.010*
20.0–29.0 23.67 ± 4.73 22.0
34.0–39.0 36.67a ± 2.52 37.0
15.0–20.0 18.0b ± 2.65 19.0
0.002*
13.0–20.0 17.33 ± 3.79 19.0
29.0–30.0 29.67a ± 0.58 30.0
10.0–12.0 11.33a,b ± 1.15 12.0
