Growth Inhibitory Effect of Lactocare on Vibrio cholerae.

BACKGROUND AND
OBJECTIVE: Acute microbial diarrheal diseases are the major public health problems in the developing countries. People affected by diarrheal diseases have the lowest financial resources and poorest hygienic facilities. Children under five, primarily in Asian and African countries, are mostly the subjects affected by microbial diseases transmitted through water.The current study aimed at investigating the comparative inhibitory effect of Lactocare (commercial probiotic) on clinical samples and standard strains of Vibrio cholerae.
METHODS: A total of 20 clinical samples and a standard strain (ATCC 14035) were provided by Health Reference Laboratory and Biotechnology Institute, respective ly. In order to confirm the samples, biochemical analysis and the polymerase chain reaction (PCR) were performed on intergenic space. Afterward, agar well diffusion method was performed in order to measure the minimum inhibitory concentration to monitor the antimicrobial activity of Lactocare.
RESULTS: Colony count of V. cholerae for the standard strain in 30% and mean for clinical samples in 50% concentration of Lactocare treatment revealed that it would propel to death phase. Since the number of colonies decreased to 100, it was considered that higher concentrations of Lactocare would completely inhibit the growth of V. cholera.
CONCLUSION: Probiotics are employed to develop new pharmaceutical preparations and functional foods in order to promote the public health.

Introduction

Accessibility to safe drinking water can significantly result in improvement of health and quality of life; therefore, efforts should be made to acquire safe drinking water. Waterborne infections are common due to the difficulty of accessing any kind of sanitary or clean water. According to the World Health Organization(WHO), about 2.5 billion people do not have access to sanitary water, and more than 1.5 million children die from diarrheal diseases each year (1). Also, it is reported that over 5 million people die per year due to water borne diseases, which more than 50% of them are microbial intestinal infections, and cholera is in the first place (2).

Probiotics plays an important role in the health of the host; a change in their balance and levels affects the health and digestion ability of the host (3). Probiotics could be employed as an effective tool for its potency to control the balance of pathogens growth, which results in the prevention of infections (4, 5). Indeed, multiple studies performed on various strains of probiotic bacteria revealed that they can participate in decreasing the number of pathogens and their virulence mechanisms (6, 7). The potency of lots of probiotic bacteria in inflammatory bowel diseases is studied in many clinical trials (8, 9). Since World War I, the non-pathogenic Escherichia coli strain is isolated and proposed for the maintenance therapy of Crohn’s disease and ulcerative colitis (9-11). Moreover, purposeful transduction of E. coli strain to newborns can prevent the colonization of the microbial intestine pathogens. The current study aimed at investigating the inhibitory effect of commercial Lactocare on Vibrio cholerae.

Materials and methods

Preparation of bacteria

The bacterial strains used in the current study were V. cholerae ATCC14035 provided by Biotechnology Institute (Iranian Research Organization for Sciences and Technology, Tehran) and 20 clinical samples provided by the Health Reference Laboratory.

Preparation of Vibrio Suspension

To prepare bacterial suspension, bacterial colo nies grown on thiosulfate-citrate-bile salts-sucrose (TCBS) agar were stirred in 1 mL phosphate-buffered saline (PBS) for 0.5 McFarland turbidity (1-1.5 × 108 colony-forming unit; CFU). In order to measure the accuracy of turbidity, samples were evacuated by spectrophotometer at 620 nm wavelength range and absorbance was set at the range 0.08-0.1 nm.

Biochemical analysis

All clinical isolates were screened by the biochemical tests including motility status using (sulfide-indole-motility) SIM agar, oxidase reaction, triple sugar iron (TSI) agar for sucrose, mannose, and arabinose fermentation tests, ornithine decarboxylase, arginine dihydrolase, and growth rate in cultures containing different amounts of NaCl (2%, 4%, 8%, and 16%) (12, 13).

DNA extraction and the polymerase chain reaction

DNA genome of each isolate was purified using a DNA extraction kit according to manufacturer’s instructions (CinnaGene, Iran) and PCR was performed according to the protocol of Chun J. et al., for 16S23S mRNA genes. The primer set was as follows: VC-Forward 5’-AGTCACTTAACCATTCAACCCG-3’ and VC-Reverse 5’-TTAAGCGTTTTCGCTGAGAATG-3’ (14, 15).

Preparation of Lactocare Suspension

The full contents of two capsules of commercial probiotics (Lactocare) purchased from ZistTakhmir Company were dissolved in 9 mL of PBS. One milliliter of the obtained suspension was added to each falcon containing MRS (de Man, Rogosa and Sharpe) broth and incubated for about 24 to 48 hours at 37°C, then centrifuged at 4000 g for 10 minutes and the supernatant was transferred under sterile conditions.

Agar well diffusion method

Agar well diffusion method was followed to determine the antimicrobial activity. Mueller-Hinton agar plates were swabbed with V. cholerae suspension and a 10-mm diameter well was punched in each center of plates. Afterward, 100 μL of Lactocare suspension was added into the well of each plate and incubated at room temperature for two hours. In collateral, 100 μL of MRS broth was fielded in case of control sample. The plates were incubated at 37°C for 24 hours for bacterial growth. Finally the inhibition zone was measured and the activity index was also calculated.

Kinetic activity of Lactocare

To obtain the kinetics of death, a certain amount of diluted bacterial and Lactocare solution was mixed in 1:1 ratio. Bacterial concentrations were defined in a manner that contained 10%, 20%, 30%, and 40% of

V. cholerae, equally well applied to the Lactocare and the remaining volume was filled up by MRS broth.

Kinetic assay and colony counting were performed at 0 to 420 minute treatment time; in collateral adding, MRS agar culture was performed in order to stimulate the optimal growth conditions of pathogenic and lactobacilli at 37°C.

Statistical Analysis

The mean ± standard deviation (SD) were calculated for clinical samples and differences between clinical samples and ATCC strain were analyzed by one-way analysis of variant (ANOVA) with SPSS version 19.0. Statistical significance was considered P <0.05.

Results

Biochemical Tests and Bacterial Culture Results

V. cholerae produced smooth yellow colonies with 2-4 mm in diameter, an opaque center, and transparent periphery on TCBS agar, after 16 to 18 hours of incubation. The results of oxidase, TSI, Voges-Proskauer (VP), motility, indole, and string of pearls test were positive. The bacteria were capable of fermenting carbohydrate on TSI with no gas or hydrogen sulfide production. V. cholerae was confirmed for all the isolates by studying the test results.

PCR assay

The PCR results revealed that all 20 clinical isolates were positive for V. chlorea (Figure 1) and the nucleotide sequence of 16S-23S mRNA genes revealed an amplicon of 300 bp.

Figure 1

Gel Electrophoresis of the PCR Products of 16S-23S mRNA Genes

Agar well diffusion method

The inhibition zone of 22 mm was significantly larger than that of CLSI guidelines (16 mm) as shown in (Figure 2).

Figure 2

Growth Inhibition Zone by Lactocare

Kinetic activity of Lactocare

V. cholerae colonies were counted and recorded in all concentrations and treatment time to obtain the kinetics of death as mentioned in Table 1.

Table 1

Number of Colonies During the Incubation With Lactocare Based on Lactocare Concentration

Time (h)
Concentration (%)	1	2			3		4	5	6		7
ATCC14035 Strain
10	2038		1950	1800		1779		1597		1521		1480
20	1424		1260	1080		972		624		552		540
30	1100		871	680		549		321		267		128
40	920		642	343		156		72		35		12
Mean Number of Clinical Isolates
10	2293		2194	1944		1913		1765		1694		1603
20	1938		1785	1597		1486		1235		1098		1076
30	1526		1428	1214		1100		883		751		717
40	1260		1143	935		816		630		517		475
50	1025		889	526		361		225		148		125
60	875		784	363		164		75		31		16

The average results from all clinical samples compared to the standard strain against each concentration of Lactocare at the incubation period is depicted in Figure 3.

Figure 3

Downward Trend of Vibrio cholerae Colonies at Various Concentrations

Statistical Analysis

A significant difference was observed between the inhibitory growth of ATCC strain and those of clinical samples at certain concentrations. As a result of the applied different treatment time and concentrations, a treatment time and concentration dependent decrease in inhibition was observed when ATCC and clinical samples were exposed to uni-colonies, where increasing treatment time resulted in reduction of bacterial growth by an average of 53% at second hour of 40% concentration for ATCC and 46% at second hour of 60% concentration for clinical samples (P ≤0.05).

Discussion

Since bacteria play an important role in the health of the host, a change in their balance and levels affects the health and ability to digest the host food. Antibiotics can be taken either directly or by consumption change in the intestinal flora and providing the pathogenic ones. On the other hand, antibiotics can increase the number of resistant bacteria, which makes treatment more difficult. Also, decrease in the levels of intestinal flora reduces the ability of carbohydrates to ferment and metabolize bile acids and cause diarrhea by absorbing a lot of water. Also, it ceases the inhibitory effect of the pathogenic bacteria growth. Finally, taking antibiotics increases the individual’s need for vitamins. Probiotics are powerful dietary supplements that help human beings to preserve the balance of beneficial gastrointestinal microorganisms in chronic conditions. Therefore, the food industry needs to carefully evaluate the safety and effects of new species and probiotic strains before combining with food products (16). Based on the European Food Safety Authority (EFSA) guidelines, microorganisms presenting minimum inhibitory concentrations (MICs) similar or less in comparison with EFSA’s breakpoint are appointed as susceptible to this antimicrobial (17).

Producing antimicrobial compounds is an important screening tool for probiotics in intestinal health; in addition to the production of metabolites such as organic acids, probiotic bacteria may produce antimicrobial agents such as bacteriocins. Probiotic bacteria may impose their effects on intestinal pathogens by reducing environmental pH. The consumed commercial Lactocare was composed of Lactobacillus casei,

L. acidophilus, L. rhamnosus, L. bulgaricus, Bifidobacterium breve, B. longum, Streptococcus thermophiles, and fructooligosaccharides (FOS) with certain colony count (10¬9 CFU) as the company claimed.

Prema et al., revealed that L. plantarum strain shows an inhibitory potency by producing bacteriocin, which was active against the indicator starins. According to Figure 2, the zone of growth inhibition was 22 mm, which was more effective in comparison with CLSI guidelines (16 mm), but could not completely inhibit the pathogen’s growth. The maximum zone of inhibition in the study by Prema was recorded about 24 mm against Salmonella typhi as an indicator strain (18); which almost confirmed the current study.

A study by Tambekar et al., used standard and commercial bacterial strains to make probiotics from cow and goat milk; the results showed that both the standard and commercial strains could tolerate the gastric acid at pH 2 and the bile salt at concentration of 2%. Tambekar et al., revealed that these bacterial strains have antibacterial activities against antibiotic-resistant enteric pathogens, which maintains their bacteriocin stability at 121°C and pH 3-9. Also, a comparative study on goat milk probiotics evolved that probiotic bacteriocins has stronger antibacterial activities versus the commercial probiotics. Although probiotic products made from domestic animals’ milk can be considered as a candida for oral therapy, the low access to such products should be considered (19).

In an in vitro study by Meshref et al., the potency of probiotic bacterial strains used for growth inhibition of E. coli, S. aureus and L. monocytogenes was reported. They also revealed that after 15 days of storage at 4°C, both L. acidophilus La-5 and B. longum ATCC15707 remained viable at >107 CFU/g, which was appropriate in yoghurt (20). The current study revealed the downward trend in the number of counted colonies at various concentrations of Lactocare versus V. cholerae in different intervals. The number of V. cholerae colonies in 30% for standard strain and mean of clinical isolates in 50% concentrations of Lactocare treatment from the first to the last hour was 103 to 102 colonies, which revealed that it could propel to death phase. According to Figure 3, since the number of colonies decreased to 100, higher concentrations of Lactocare would completely inhibit the growth of V. cholera.

Conclusion

Probiotics are employed to develop new pharmaceutical preparations and functional foods in order to promote the public health.