Antagonistic Effects of Lactic Acid Bacteria Isolated from the Feces of Breastfed Infants Against Enterotoxigenic and Enteropathogenic Escherichia Coli

Antagonistic Effects of Lactic Acid Bacteria Isolated from the Feces of Breastfed Infants Against Enterotoxigenic and Enteropathogenic Escherichia coli

Zahra Alipoor 1, Zoheir Heshmatipour*1and Shaghayegh Anvari 2

1Department of Microbiology, Islamic Azad University, Tonekabon Branch, Tonekabon, Iran. 2Department of Bacteriology, Golestan university of Medical Sciences, Golestan, Iran Received: May 23, 2015 Accepted: September 1, 2015 ABSTRACT

The purpose of this work was to evaluate the antagonistic characteristics of probiotic lactic acid bacteria (LAB) from the feces of breast-fed infants against enteric pathogens Enterotoxigenic Escherichia coli (ETEC) and Enteropathogenic Escherichia coli (EPEC) which cause diarrhea in newborns and children. LAB strains were isolated from the feces of 6- 12 month- old breast-fed infants in MRS and M17 media and were identified by using the morphological, phenotypical, biochemical and 16SrRNA gene sequencing method. Finally, the antagonistic effect of these isolates against ETEC and EPEC was assayed by the well diffusion test. 15 isolates showed the primary characteristics of Lactic Acid Bacteria. We randomly selected three colonies out of these 15 isolates for Some probiotic potential tests and 16SrRNA gene sequencing method. The selected isolates, were found Enterococcus raffinosus, Enterococcus sp. PFC 4 and Enterococcus faecium , and all of them showed good antagonistic effects against ETEC and EPEC. These results proved the effectiveness of antagonistic active compounds produced by LAB against enteric pathogens like: EPEC and ETEC. KEYWORDS: Enterophatogenic Escherichia coli , Enterotoxigenic Escherichia coli, Lactic acid bacteria, infant feces and antagonistic effects.

INRTODUCTION

Enterotoxigenic Escherichia coli is an important cause of acute diarrhea in newborns, young kids and travelers [1,2,3]. The major virulence factors of ETEC that causes watery diarrhea are heat labile toxin (LT) and heat stable toxin (ST) [4, 5]. The percentage of cases of sporadic endemic infant diarrhea, which are due to ETEC Usually vary from 10 to 30% [6]. Enteropathogenic Escherichia coli (EPEC) strains are the major cause of diarrhea among infants in developing countries. The clinical characteristics of EPEC are watery diarrhea, vomiting and low-grade of fever [7-8]. The term probiotic refers to a living non-pathogenic microorganism which, when administrated in a sufficient number, confer a health benefit on the host [9]. Several factors should be considered to identify the effective probiotic bacteria. They should be of human origin and also have the capacity to survive in the gastrointestinal tract [10]. They also should possess some properties such as: resistance to acidic pH, resistance to bile salt concentration and adherence to epithelial cells in the intestine [11]. Among the usually used microorganism; Lactic acid bacteria (LAB) are regarded as a major group of probiotic bacteria [12-13]. Most probiotic microorganisms belong to LAB. Such as: Lactobacillus sp , Streptococcus sp and Enterococcus sp [14]. Human milk is a rich source of LAB for the infant gut [15]. Lactic acid bacteria transfer to the infant intestine from mother's milk and colonizes in the gastrointestinal tract. There is some evidence that proves the efficacy of LAB for both prevention and treatment of acute diarrhea [16]. LAB can probably reduce diarrhea in several ways, such as competing with pathogens for nutrients and space in the intestines, producing organic acid, bacteriocins, antibiotic-like substances and stimulating the immune [17]. Enterococcus species are used as probiotic Lactic acid bacteria to improve the microbial balance of the intestine and treat gastroenteritis in human and animal [18]. They are gram positive, catalase negative and non- spore forming cocci that appear in pair or short chain, facultative anaerobes and tolerant of extreme temperature [19]. There is some experimental proof that the fecal microflora in infants may contain species, including Enterococcus, Lactobacillus, Streptococcus and Bifidobacterium that produce antimicrobial substances against other intestinal bacteria [15]. The16SrRNA gene containing variable and conservative regions in the nucleotide sequence, Which is the most desirable form of the determination of genus and Species affiliation of a bacterium [20]. Thus, the Sequencing analysis of the 16s RNA gene is used in this study to confirm and diagnose of the isolated LAB at the genus and species level.

*Corresponding author: Zoheir Heshmatipour, Department of Microbiology, Islamic Azad University, Tonekabon Branch, Tonekabon, Iran. Email: [email protected] Telephone: +98 9112911915

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MATERIAL AND METHODS

Fecal sample preparation and isolation of LAB Fresh fecal samples were obtained from 20 healthy breast-fed Iranian infants aged from 6-12 month. They were collected in sterilized plastic bags early in the morning, and were transferred to the microbiology laboratory at Azad university of Tonekabon. 1g of samples were completely mixed with 9 ml of distilled water and serially diluted (10 -1- 10 -5). Then, 100μl of the two final tubes with the least dilution (10 -4-10 -5) was cultured on MRS and M17 agar (Company of Merk, Germany) and incubated anaerobically at 37 0C for 48 hours [21].

Identification of LAB 30 colonies were isolated from20 samples. The isolates were assayed by some conventional test like: Gram staining, catalase reaction and motility test. 15 isolate showed good primary characteristics of LAB. Out of these 15 colonies; 3 of them were selected randomly for probiotic potential tests such as: acid tolerance, bile salt resistance and carbohydrate fermentation [22].

Acid tolerance In this study, resistance of selected isolates to acidic pH (3- 4-5-6) was evaluated. 100μl of the 24 hour cultures of the isolates was inoculated to MRS and M17 broth media that adjusted to acidic pH and incubated for 24 hours at 37°C. Then, the survival percentage of strains to acidic pH was determined by plating serial dilution on MRS and M17 agar. After 24h, the presence of colonies on agar media was evaluated [21].

Bile salt tolerance The resistance of selected isolates to Sodium deoxycholate salt was determined by broth assay. 100μl of overnight cultures of isolates was added to 5ml MRS and M17 broth containing 0.3% sodium deoxycholate salt and incubated anaerobically at 37°C. Then, Culture's turbidity was monitored spectrophotometrically (650nm) for growth at time intervals of 0,15-40 (minute), 1h and 24h after incubation. The positive control contained MRS and M17 broth without bile, and the negative control had MRS and M17 media without any bacteria. The experiments were conducted in triplicate [23].

Carbohydrate fermentation test (API Test): 3 selected isolate were assayed for their carbohydrate's fermentation profile. Moreover, the isolates were examined for their ability to ferment sugars by using API20STREP kit. The results of the fermentation profiles were compared with the information from the computer-aided software of the API Kit [22].

DNA extraction and PCR of 16srRNA gene 1.5 ml of 24h broth culture of selected isolates, was transferred to a micro centrifuge tube and centrifuged at 3,000 rpm for 8min. The supernatant was discarded. 100μl of 0.5% lysozyme and 75μl of 10% SDS was added to the tubes. It was mixed well and incubated for 1h at 37°C. After the incubation, a same volume of phenol: chloroform (25:24) mixture was added, and mixed well by inverting the tubes gently until the phases were completely mixed and centrifuged at 10, 000rpm for15 min. After centrifugation, the upper aqueous phase was transferred to a new tube and equal volume of chloroform was added. Then, it was centrifuged at 10,000 rpm for 15min, the gained upper sedimentary phase was transferred to a new tube and 0.6 volumes of isopropanol was added and mixed gently and incubated for 30 min in -20°C until the DNA gets precipitated, and subsequently centrifuged at 13,000 rpm for 15 min. 70% ethanol was added to the pellet and centrifuged at 14, 00rpm for 1min. 30μl of injected distilled water was added to the pellet and stored in -20°C [24]. Finally, the quality of the extracted DNA was evaluated by gel electrophoresis and investigation of optical density at 260 and 280nm. The PCR reaction mixture (25μl) contained: 5μl of template DNA, 1μl (10 pMol) of forward primer F-5'- AGACTTTGATCCTGGCTCAG-3', 1μl of Reverse primer R-5'GGTTACCTTACT-3', 10μl of the PCR master mix (TaqDNA Polymerase2.0, 1.5mMmgcl 2, Ampliqon company, Denmark) and 8μl of Distilled water. The reaction was performed with initial heating at 95°C for 5 min, 30 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 75s, extension at 72°C for 75s, followed by a final extension at 72°C for 10 min. 5μl of PCR product was run on agarose gel electrophoresis along with the DNA molecular weight marker (1Kb DNA Ladder, 250-10000bp, Cinagen, Iran) to analyze the product. PCR products were sequenced by the Genfanavaran company in Iran and results of sequencing was compared with the NCBI database using the BLAST search available through NCBI website.

Investigation of antagonistic characteristics in LAB In order to detect antagonistic effects of LAB against reference strains ETEC (ATCC 35401) and EPEC (ATCC 43887), The selected isolates were investigated by the well - diffusion method. In this test 5μL of the overnight cultures of enteric pathogens ETEC and EPEC were cultured in the Muller Hinton agar plates, and wells were made in the agar with a sterile glass rod. Then, 100μL of the LAB supernatants obtained from the 24, 48 and 72h cultures was dropped into the well. Plates were incubated for 24 h at 37C [25].

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RESULTS

Fecal sample preparation and isolation of LAB Out of 30colonies, 15 isolates were Gram-positive in the form of bacill, coccobacilli and cocci, catalase and motility negative. These results showed that the isolates had the primary characteristics of LAB.

Acid tolerance Results indicated that the selected isolates were resistant to different acidic pH, and the maximum growth ratio for all species was obtained at pH 6.0 and 7.0. The least growth ratio observed at pH 3. The species Enterococcus sp. PFC4 didn't show growth at pH 3.0.

Bile salt tolerance Results showed that (Figure1) the resistance of selected isolates to 0. 3% sodium salt has developed by increasing the incubation time. The greatest resistance being obtained for the Enterococcus raffinosus . The most sensitive species to bile salt was Enterococcus faecium . In this assay, the greatest growth of LAB in the presence of bile salt was occurred after 24-hour incubation (Figure1).

Figure1:Diagram of Bile salt resistance in Lactic Acid Bacteria

Carbohydrate fermentation test (API): According to the Results of Carbohydrate fermentation (Table1), selected isolates were identified: Enterococcus faecium, Enterococcus raffinosus and Enterococcus sp. PFC 4.

Table 1. Carbohydrate fermentation profiles of isolated Lactic Acid Bacteria Carbohydrate fermentation profiles GLYG AM R R A M S L T I R Identified strains D A I R A O A R N A F B A N R C E U F - - - + + + - + + - - Enterococcus faecium - - - - - + + + + + - - Enterococcus sp. PFC4 - - - + - - - + + - - Enterococcus raffinosus

RAF: Rafffinose, INU :Inulin, TRE:Trehalose, LAC: Lactose, SOR: Sorbitol, MAN: Mannitol, ARA: Arabinose, RIB: Ribose, AMD: Amidon ,GLYG: Glycogen

DNA extraction and Polymerase chain reaction The genomic DNA of the isolated species was extracted, and DNA samples were observed on agarose gel electrophoresis. The optical density ratio of the extracted DNA in 260 and 280nm was 1.8. And it showed the suitable purity of the DNA samples for PCR reactions. The PCR product electrophoresis results showed that the isolated strains were positive for 16SrRNA gene, and they were observed in1500bp region. Sequencing results of 16SrRNA were

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exported to the NCBI database and checked for homology alignment. Based on the alignment results, the selected isolated were found as Enterococcus faecium , Enterococcus raffinosus and Enterococcus sp. PFC4 (Figure 2).

Figure 2: electrophoresis results of Isolated LAB. 1: Negative control 2: lane of Enterococcus faecium M: 1Kb Ladder. 3: lane of Enterococcus raffinosus 4: lane of Enterococcus sp. PFC4

Antagonistic characteristics in LAB The zones of inhibition in well diffusion method demonstrated that the isolated species, Enterococcus faecium , Enterococcus raffinosus and Enterococcus sp. PFC4 have antagonistic activity against enteric pathogens ETEC (ATCC 35401) and EPEC (ATCC43887). The diameters of inhibition zones were then measured. Enterococcus faecium showed strong activity against ETEC with the inhibition zone of 14 mm in diameter, and EPEC was moderately inhibited by Enterococcus sp. PFC4 . (Table 2). The greatest antagonistic activity of isolated LAB against pathogenic strains was observed after48h incubation of LAB supernatants (Figure3).

Table 2. Diameter of the zone of inhibition (mm) produced by Lactic Acid Bacteria on enteric. Pathogens ETEC and EPEC by using the well diffusion assay. LAB strains Photogenic Enterococcus Enterococcus sp. Enterococcus strains raffinosus PFC4 faecium ETEC 10 10 14 EPEC 10 9 11

Figure3: Inhibition zones of antagonistic activity in 3 selected isolate: Enterococcus Faecium , Enterococcus sp. PFC4 and Enterococcus raffinosus on A) EPEC B) ETEC In figure A: number of 12 is Enterococcus faecium; number of 10 is Enterococcus sp PFC4 and number 8 is Enterococcus raffinosus In figure B: number of 6 is Enterococcus faecium; number of 2 is Enterococcus raffinosus and number of 3 is Enterococcus sp PFC4.

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DISCUSSION

LAB strains are the most common microbes which are regarded as probiotic [26, 27]. They have the ability to colonize and proliferate in the intestine tract by helping to prevent Ecoli, Salmonella. Spp and other pathogenic bacteria from adhering to the intestinal cells [28]. In this study LAB, strains were isolated from 20 breast-fed infant feces. According to the result of phenotypical identification tests, the isolates were gram positive, in the form of bacill, cocobacill and cocci, catalase and motility negative. The traditional identification of bacteria based on phenotypic characteristics has some limitations. And it is not as accurate as the identification based on genotypic methods [29]. Therefor, in this study, molecular analysis has proven to be the most reliable method for the exact identification of lactic acid bacteria The16S rRNA gene sequence is the most suitable form for the determination of genus and species affiliation of a bacterium [20]. Therefore, in this study confirmation of the isolates was performed by 16SrRNA gene sequencing. According to API and PCR tests, isolates were identified Enterococcus faecium , Enterococcus raffinosus and Enterococcus. Sp.pFC4. Boycheva et al and Huidrom et al also identified enterococci species such as: E. faecalis and E. faecium in infant feces [30, 31]. The isolate Enterococcus faecium was the predominant species in this survey. This result was similar to the finding of Wang et al [32]. In other studies, strain's Lactobacillus acidophilus , [31] lactobacillus plantarum [22] and Lactococcus lactis [21] were the most predominant species in infant feces. In order to evaluate beneficial effects of lactic acid bacteria in the host, these probiotic bacteria must pass through the highly acidic stomach condition in order to reach the intestine [33]. Thus, resistance to low pH is essential for the survival of LAB in the gastrointestinal tract. Results showed that the isolated LAB were resistant to different pH and grew well in acidic pH. This event proves the ability of the LAB to survive in different sections of the gastrointestinal tract with different pH. In this study, the maximum growth ratio for our species was obtained at pH 6, and the least growth was observed at pH 3. This result was similar to Plinescue et al [21]. Resistance to bile salts is generally considered as an essential characteristic for probiotic strains to survive the conditions in the small intestine. The presence of bile salts in the environment of bacterial culture is much more harmful than the effect of low pH [22]. The average concentration of bile salts in the gastrointestinal tract is believed to be 0/3% [34]. Because of this event, in this study, 0.3% sodium deoxy cholate was used and resistance of strains to bile salt was assayed in different incubation time. The result showed that the increment of incubation times had the positive effects on bile salt resistance of species, And the isolates had the most resistance after 24h incubation. Khalill et al used a variable concentration of bile salt in a stable incubation time for bile salt resistant assay, and they could prove the resistance of isolated LAB to different concentrations of bile salts [22]. EPEC and ETEC are two significant categories of Ecoli, which have been linked to infant diarrhea in developing countries, on the other hand, the percentage of diarrhea incidence in breast –fed infants are lower than formula-fed., And this event is because of LAB colonization in an infant's gastrointestinal tract that has inhibitory activities against enteropathogen. LAB inhibit enteropathogen by the secretion of antimicrobial substance such as organic acids (lactic acid, acetic acid, formic acid), other sugar catabolites (ethanol, diacetyl, carbon dioxide), bacteriocins, oxygen catabolites (hydrogen peroxide), antibiotic-like substances (reuterin and reutericyclin) and others [35]. In this research, the antagonistic effect of LAB was tested by the well diffusion method. Results showed that all enterococci strains had a good antagonistic effect against ETEC and EPEC. Similar results have been reported by Boycheva et al for Enterococcus faecalis and Enterococcus faecium against different Ecoli strains [30]. In this research the species Enterococcus faecium had the most antagonistic activity against Ecoli strains with the inhibition zone of 11 and 14 mm. These results were similar to Huidrom et al [31]. In this study the greatest antagonistic activity of isolated LAB against pathogenic strains was observed after48h incubation of their supernatants. This result was similar to compose et al [36]. They have reported that this action is probably due to production of organic acids and bacteriocins. Bacteriocin is one of the antimicrobial substances that optimally produce between 48 and 60h [37]. Compos et al also declared that the maximum production of bacteriocin from the selected LAB strains is observed in the stationary phase of growth. Which ranges from 21 to 72hour. Therefore, these reports prove the reason for maximum inhibitory activities of our isolated LAB after 48h incubation.

CONCLUSIONS

We can conclude from this study that antagonistic active compounds which produced by LAB are effective at EPEC and ETEC. These results prove the potential of isolated LAB to inhibit enteropathogens like: and ETEC. Therefore, they can be used in the treatment of infantile diarrhea, which is caused by EPEC and ETEC.

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ACKNOWLEDGEMENTS

The authors are very grateful to all members of the microbiology research laboratory in Azad university of Tonekabon, Iran, who helped them in project performance.

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