Isolation and Characterization of Lactic Acid Bacteria Producing Bacteriocin from Newborn Infants Feces

Abstract

Ninety-six strains of bacteria were isolated from newborn infants feces. After Antimicrobial activity was screened, these strains showed a broad inhibition spectrum. Particularly, twenty-one strains had antimicrobial abilities to inhibit Escherichia coli ATCC 35150, Micrococcus luteus CICC 10209, Salmonella typhimurium ZJJK 311. Regarding the physiological analysis of strains and BLAST analysis of 16S rDNA sequences, twenty-one strains were divided into six species: Enterococcus faecium (six isolates), Enterococcus faecalis (six isolates), Enterococcus durans (two isolates), Lactobacillus paracasei (one isolate), Lactobacillus rhamnosus (three isolates), and Lactobacillus plantarum (three isolates). All of the selected strains showed antimicrobial activity against six food-borne pathogens. After treatments with proteinase K or Trypsin, L. paracasei (LZ54), Enterococcus faecalis (LZ95), and L. plantarum (LZ222, LZ227) lost antibacterial activity, indicating that the proteinaceous nature of the antimicrobial compounds were produced by these strains. These strains could be introduced as preservatives in the food industry.

Keywords: Lactic acid bacteria; Bacteriocin; Antibacterial

Introduction

The lactic acid bacteria (LAB) constitute a large group of nonsporulating, gram-positive, catalase and oxidize-negative rods and cocci. These bacteria produce lactic acid as the major metabolite of the carbohydrate fermentation. All LAB are anaerobic and aero tolerant. They generally have complex nutritional requirements. The main genera of the LAB are Lactobacillus, Lactococcus, Streptococcus, Enterococcus, and Sporolactobacillus. LAB plays an important role in physiological functions and exists widely in the intestinal tracts of the human body. Lactobacilli, Bifidobacteria, and Enterococci are normally associated with infant gut microbiota [1].

In neonates, especially preterm babies, intestinal microbiota plays important roles in metabolism, nutrition, immune functions, and defense against pathogens. However, many of the microfloras that colonize the neonatal gut have different origins. Recent molecular studies have shown that LAB colonization is not significantly related to the method of delivery. Rather, it is also influenced by the infant diet during the first few days of life [2]. Formula-fed infants have greater numbers of more diverse enterobacteria and enterococci and maintain these populations longer [3, 4]. In contrast, breast milk seems to facilitate the growth of maternal lactobacilli in the infant gut [2, 5].

Recent research has revealed that the probiotic effects of LAB including inhibiting the growth of pathogenic bacteria, reduce therate of colon cancer, increase the immune response, and decrease serum cholesterol [6, 7, 8]. It has also been reported that they produce antimicrobial substances, including bacteriocins, organic acids, and low-molecular-weight substances that can inhibit the growth of undesirable pathogens that can cause diarrhea or other diseases of the human intestine [9, 10, 11]. We choose the feces of healthy newborn babies from Zhejiang Maternal and Child Health Hospital as the research objects for screening the strains capable of producing antibacterial substances. Special attention was paid to bacteriocins.

The aim of the present work was to characterize and identify the lactic acid bacteria isolated from feces of healthy newborn babies in Zhejiang Maternal and Child Health Hospital and to select bacteriocins-producing strains for use in the food industry.

Materials and Methods

Sample collection

Three hundred and forty-seven samples were collected from newborn infants in Zhejiang Maternal and Child Health Hospital. These infants defecated 72 h after birth, generally three or four pieces per infant. The samples were placed at 4 °C as soon as they were collected, during transporting way, and at the laboratory. After examination, they were disposed of immediately.

Bacterial strains

Lactic acid bacteria (LAB) were isolated from samples of newborn infants. Standard ten-fold dilutions of the feces were made in 0.9 % sterile physiological saline, and each sample was plated on both MRS (Becton Dickinson, U.S.) agar and M17 (Becton Dickinson, U.S.) agar containing CaCO3 (5 g/L). A volume of 0.1 mL of appropriate dilutions was spread and plated in triplicate on the following medium for isolation of LAB: M17 agar for lactococci and MRS agar for lactobacilli. All plates were incubated at 30 °C under anaerobic condition until colonies became visible. Colonies were randomly selected based on morphological differences (colony size and shape, clearance zone formed from hydrolysis of CaCO3 by lactic acid). Colonies were purified, again on the MRS and M17 agar plates. The isolates were routinely maintained in broth at 30 °C and stock culture of each isolate was kept for further study at -80 °C in broth supplemented with 30 % glycerol.

Indicator bacteria used for antimicrobial assays were cultured in Luria-Bertani (LB) agar under aerobic conditions at 37°C. These bacteria included Escherichia coli ATCC 35150, Micrococcus luteus CICC 10209, Staphylococcus citreus ZJJK 311, Vibrio parahaemolyticus ATCC 27519, Salmonella typhimurium CGMCC 1.1190, and Listeria monocytogenes ATCC 7644. These strains were provided by Microbiological Laboratory of Clinical Detection Center of Zhejiang (Hangzhou, China) or from our own supplies.

Antimicrobial assays

Cell-free culture supernatants (CFCS) were obtained by centrifugation (12,000×g, 4°C, 20 min) of LAB cultures grown in 20 mL broth at 30 °C for 24 h. The supernatant was filtered through a 0.22 µm filter to remove residual cells. The antimicrobial activity of all samples was tested using the agar-well assay [12]. Briefly, indicator bacteria were grown in LB broth overnight and spread onto the soft agar (0.75 %, w/v) plate of LB after diluting to 107 CFU/mL. Then wells 8 mm in diameter were punched onto the surface using a sterile borer. Then 200 µL sample prepared from filtrate was added to each well of the plate and stored at 4 °C for 30 min. Samples were then incubated at 37 °C for 18 h. Antimicrobial activity was recorded as growth-free inhibition zones (diameter) around the well. MRS and M17 adjusted to 4.5 as controls.

Sensitivity to proteolysis enzymes of the cell-free supernatants of bacteriocins producer strains was assessed through treatment with proteinase K and Trypsin (Sangon). All enzymes (10 mg/mL in sterile distilled water) were filter-sterilized and added to supernatants at a final concentration of 1 mg/mL in phosphate buffer (pH 6.5). Following incubation at 37 °C for 2 h, enzymes were denatured by heating at 100 °C for 5 min. Untreated samples were used as controls. The residual activity of enzyme-treated samples against Listeria monocytogenes was determined using the well-diffusion method [12].

The effect of temperature on cell-free supernatants was determined by treatment at 80 °C or 100 °C for 20 min in water bath and at 121 °C for 20 min (sterilization). A portion of each supernatant was treated at room temperature for 20 min. This served as a control. Samples retaining activity after heat treatment were identified through the well-diffusion method [12]. Regarding our previous work, Gramnegative bacteria Escherichia coli and Gram-positive bacteria Micrococcus luteus are more sensitive to bacteriocins. In this work, Escherichia coli ATCC 35150 and Micrococcus luteus CICC10209 were chosen as the indicator strains.

Biochemical characterization

Each isolated strain was propagated twice on a plate before use. We used bacterial trace biochemical reaction tubes produced by Hangzhou Microbial Reagent Co., Ltd.

Identification of LAB isolates

Identification of the twenty-one LAB isolates was performed using Phylogenetics analysis based on 16S rRNA sequence.

Total genomic DNA from each isolate was extracted from 3 ml pure overnight cultures grown in broth at 30 °C using a modified version of the CTAB (cetyltrimethylammonium bromide) method [13, 14]. Purified DNA was diluted to 100 ng/µL for further use.

Genomic DNA from each isolate was used as a template for PCR amplification of a segment of the 16S rDNA gene. Genomic DNA from each isolate was used as template for PCR amplification of 16S rRNA gene using the primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-CTACGGCTACCTTGTTACGA-3') [15, 16]. The 25 µL reaction mixture contained 100 ng templates DNA, 2.5 µL 10×buffer, 1 µL 3 mM MgCl2, 1 µL 0.2 mM of dNTP, 0.5 µL 10 pmol of each primer, and 6.0 units of Taq DNA polymerase. PCR amplifying procedures were as follows: 5 min at 94°C, 30 cycles of 1 min at 94°C, 30 s at 54°C, 1 min at 72°C, and then 7 min at 72°C. It was carried out in an automatic thermal cycler. The sequencing of purified products was performed by Shanghai Sangon Biosciences Corporation of China (Shanghai, China).

Construction of phylogenetics trees

The 16S rDNA gene sequences of all twenty-one isolates were submitted to the National Center for Biotechnology Information (NCBI, https://blast.ncbi.nlm.nih.gov) for BLAST search. The sequences of four bacteriocins-producing strains were imported into MEGA version 4.0 software, with which a sequence alignment and Phylogenetics tree were created on the basis of the neighbor-joining method [17]. The reliability of the groups was evaluated by bootstrap analysis with 500 resamplings. Bacillus subtilis DSM 10T was used as an outgroup.

Results

Detection of antimicrobial activity

Twenty-one isolates collected from infant feces were isolated on plates and screened for antimicrobial activity against Escherichia coli ATCC 35150 and Micrococcus luteus CICC10209 using ager well diffusion assay. Then the CFCS of the 21 isolates were analyzed and found to show wide-spectrum antibacterial activity, some of them can inhibit all the indicate bacteria in our study, including Staphylococcus citreus ZJJK 311, Vibrio parahaemolyticus ATCC 27519, Salmonella typhimuriumCGMCC 1.1190, and Listeria monocytogenes ATCC 7644. These isolates displayed antibacterial effects against at least three indicator strains (Table 1).