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Park, Song, Shin, Lee, Park, Song, Lee, Lee, Seo, Kim, Lee, Kim, and Choi: Investigation of antibacterial activity of Oenothera biennis L. extract against Salmonella Typhimurium

Abstract

Salmonella species is one of the major bacterial agents that causes gastrointestinal disease in pigs. Natural antimicrobials derived from plants may be alternative therapeutics that could replace currently used antibiotics in the control of infectious disease. In this study, we assessed the antibacterial activity of Oenothera biennis L. extract against Salmonella Typhimurium both in vitro and in vivo. O. biennis L. extract had a strong inhibitory effect on S. Typhimurium in vitro, reducing bacterial growth by 87%. For the in vivo experiment, 16 post-weaned pigs were divided into 4 groups consisting of 4 pigs each: an uninfected, untreated negative control group; an untreated positive control group that was infected with S. Typhimurium; and two groups that were infected with S. Typhimurium and treated with either 0.1% or 0.5% O. biennis L. extract. Pigs were followed for 21 days after infection, and their body weight, daily gains, feed conversion ratio (FCR), and feed efficiency (FE) were monitored. The pigs treated with O. biennis L. extract had significantly higher daily gains than the positive control group (p<0.05). The O. biennis L. extract-treated pigs also exhibited better weight gain and FE, as well as a lower FCR and less severe diarrhea, than the positive controls but these results were not statistically significant. Our study demonstrates that O. biennis L. extract has antibacterial effects against S. Typhimurium, both in vitro and in vivo, and that these antibacterial effects may produce better growth performance in pigs infected with S. Typhimurium.

INTRODUCTION

Oenothera biennis L., a member of the Onagraceae family, was first grown by North American [6]. Native Americans used folk drugs containing extracts of O. biennis L. to treat a number of ailments, including inflammation, rash and cough [23, 25]. O. biennis L. contains ellagitannin dimers, formed by oenothein, that have anti-cancer and antibacterial activity [11, 17, 20, 21]. In addition, MTT analysis has shown that some phenolic compounds extracted from O. biennis L. are cytotoxic and selectively induce apoptosis of tumor cells [15]. Salmonella species are significant zoonotic pathogens, causing infectious disease in mammals, reptiles, birds and other animals following the ingestion of food contaminated with fecal matter. Indeed, Salmonella infection is one of the leading causes of food poisoning in Korea; the major symptoms of salmonellosis include septicemia, food poisoning, pyrexia and diarrhea [2]. Salmonella are gram-negative bacteria, with various serotypes and a wide host range, making these species some of the most common enteric bacteria.
Infection with Salmonella is considerably difficult to treat, due to the various serotypes of the bacteria and the extended time of infection in host cells [3]. Antibiotics have thus been widely used to prevent Salmonella-related disease, as well as to promote growth and improve feed efficiency (FE) in farmed livestock. However, the excessive use of antibiotics has produced adverse effects, including the emergence of drug-resistant bacteria and the presence of antibiotic residue in livestock products [12]. To combat these problems, the development of antibiotic agents derived from natural substances has been investigated in studies conducted both domestically and overseas [16]. In this study, we analyzed the antibacterial effect of O. biennis L. extract on Salmonella both in vitro and in vivo.

MATERIALS AND METHODS

Bacterial strains and culture medium

Salmonella Choleraesuis (ATCC 13312), S. Enterica (ATCC 43971), and S. Gallinarum (ATCC 9184) were provided by the American Type Culture Collection (ATCC, USA), while S. Typhimurium (KCCM 11862) and S. Enteritidis (KCCM 12021) were provided by the Korean Culture Center of Microorganisms (KCCM, Korea). All Salmonella species were grown at 37 °C in nutrient broth (Difco, Detroit, MI, USA) [7].

Preparation of O. biennis L. extract

After air-drying at room temperature, O. biennis L. samples were ground to powder using a mechanical grinder. Approximately 100 g of the powder was extracted by stirring continuously in 2,000 mL ethanol, at 100 rpm for 24 hours at room temperature. The extracts were filtered, followed by removal of the solvent in a rotary vacuum evaporator (N-21NS, EYELA, Tokyo, Japan). Extracts were subsequently freeze-dried and stored at −20 °C until required for testing.

Determination of the minimal inhibitory concentration of O. biennis L. extracts

Minimal inhibitory concentration (MIC) was determined by the micro-dilution method, using two-fold serial dilutions of the plant extracts, according to the NCCLS [13]. These serial dilutions yielded a final extract concentration range of 0.039 to 5 mg/mL. Microtiter plate wells were then filled with 100 μL of each dilution of extract; wells containing 100 μL of Mueller Hinton broth without extract were included as negative controls. Bacterial inocula were adjusted to contain approximately 1 × 105 CFU/mL and test plates were incubated at 37 °C for 24 hours.

Growth inhibition of microorganisms by O. biennis L. extracts

To determine the inhibition of growth of various Salmonella serotypes by the plant extracts, 1 × 105 CFU/mL of bacteria were inoculated into 10 mL of nutrient broth containing 0.16 mg/mL of extract; cultures were then incubated at 37 °C for 24 h. Following incubation, the growth media was spread onto nutrient agar plates and the resulting number CFU was counted. The rate of inhibition of bacterial growth was measured by comparing the CFU of the treated samples to the CFU of the negative control.

Antibacterial activity of O. biennis L. extracts in vivo

The in vivo experiments were performed at a general pig farm located in Pyeongtaek-si, Gyeonggi-do, Korea. Sixteen weaned piglets, weighing 6–8 kg each, were assigned to four groups. Piglets in the two treated groups received either 0.1% or 0.5% O. biennis L. extract, while animals in the positive and negative control groups received no extract. Three days after treatment, the positive control, 0.1% extract-treated, and 0.5% extract-treated piglets were challenged orally with 5 × 108 CFU/mL of S. Typhimurium; the negative control animals were not infected. The piglets’ body weight, FE, feed conversion ratio (FCR), average daily gains (ADG) and fecal signs were subsequently evaluated [18].

Statistical analyses

Statistical significance of the experimental results was analyzed by one-way ANOVA and ANCOVA using the SPSS Statistics ver.19 (USA) program [14]. Data are expressed as mean ± SE.

RESULTS

Determination of the MIC of O. biennis L. extract

The micro-dilution method was used to identify the antibacterial effect of O. biennis L. extract against five different serotypes of Salmonella. No inhibition of bacterial growth was found when the test strains were treated with 50% DMSO (negative control). However, concentrations of O. biennis L. extract >0.16 mg/mL exhibited antibacterial activity against all Salmonella strains tested (Table 1).

Inhibition of Salmonella growth by O. biennis L. extract

O. biennis L. extract was also able to inhibit the growth of the Salmonella species examined in this study. The extract inhibited bacterial growth rate by 35% for S. Choleraesuis, 38% for S. Enterica, 58% for S. Enteritidis, and 24% for S. Gallinarum. The extract had a particularly strong effect on S. Typhimurium, inhibiting the growth rate of this species by 87% (Fig. 1).

Antibacterial activity of O. biennis L. extract in vivo

The O. biennis L. extract also demonstrated considerable inhibition of S. Typhimurium growth in the in vivo antibacterial assays performed in pigs. Piglets treated with either 0.1% or 0.5% O. biennis L. extract had a higher survival rate than the positive control group, where two of the four piglets succumbed to infection. Piglets in the negative and positive control groups had an increase in average body weight of 3.5 and 3.3 kg, respectively, over 21 days. However, piglets treated with either 0.1% or 0.5% O. biennis L. extract had an increase in body weight of 5.1 and 6.2 kg, respectively, over the same duration. While piglets treated with O. biennis L. extract showed a higher rate of body weight increase over 21 days than the two surviving piglets in the positive control group, the result was not statistically significant (Fig. 2).
ADG of piglets treated with 0.1% or 0.5% extracts of O. biennis L. extract was 0.81 and 0.99 kg/day, respectively: a significant increase compared to the positive control group (p<0.05) (Fig. 3).
In the negative and positive control groups, the FCR was 1.34 and 1.95 kg/day, respectively, over the course of 21 days. In contrast, the FCR of piglets treated with 0.1% or 0.5% O. biennis L. extract was 1.15 and 1.44 kg/day, respectively, for 21 days. FCR values for the piglets treated with either 0.1% or 0.5% extract were not significantly lower than those of the positive control group (Fig. 4). Similarly, piglets treated with O. biennis L. extract also showed higher FE over the course of 21 days than the two surviving piglets in the positive control group, but these results were not statistically significant (Fig. 5).
Piglets in the positive control group had signs of diarrhea for 7 days after challenge with S. Typhimurium. Piglets treated with either 0.1% or 0.5% O. biennis L. extract, however, had signs of diarrhea for only 3 days after challenge. Piglets in the negative, uninfected, control group had normal fecal excretion for the duration of the study (Table 2).

DISCUSSION

As of 2011, as prohibitions increase on the addition of antibiotics to animal feed, the frequency of bacterial diseases, such as salmonellosis, is predicted to rise. However, many studies have investigated the use of alternative antibiotic agents such as egg yolk [1, 10] and probiotics [19]. There have also been a number of recent studies assessing the antibiotic properties of plant extracts [8, 9, 24], with common plants such as garlic, clove, guava, and lemongrass have showing potential as antibiotic agents, both in vitro and in vivo [5]. Choi et al. reported that treatment with punica granatum peel extract resulted in decreased bacterial shedding and mortality during Salmonella infection of mice and pigs [4]. Tuner et al. demonstrated effective results on ADG and FCR when weaned pigs were infected with S. Typhimurium and fed Quillaja saponaria for 24 days, but these results were not statistically significant [22].
A study by Hammer et al. found that O. biennis L. seed had no antibacterial activity against E. coli, Acinetobacter, Enterococcus, Klebsiella and S. Typhimurium in vitro [5]. However, our results suggest that the aerial portion of O. biennis L. has antibacterial activity and can inhibit the growth of S. Typhimurium. Notably, we found significant increases in the ADG of S. Typhimurium-infected piglets treated with O. biennis L. extract. The body weight and FE values for infected piglets treated with O. biennis L. extract also increased more than the infected, untreated piglets in the positive control group. Additionally, piglets treated with O. biennis L. extract had a faster recovery, based on fecal signs, than the positive control animals. Overall, our results indicate that extract of O. biennis L. may relieve salmonellosis in vivo and suggest that adding O. biennis L. extract pig feed may decrease Salmonella-related economic damage to the pig industry in Korea.

Acknowledgments

This study was supported by Rural Development Administration (RDA) (PJ007611).

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Fig. 1
Inhibition efficiency of Oenothera biennis L. extract on the growth of S. Choleraesuis, S. Enterica, S. Typhimurium, S. Enteritidis and S. Gallinarum.
jpvm-38-1f1.tif
Fig. 2
Body weights of pigs in 4 groups. Negative control group was neither treated with Oenothera biennis L. extract nor infected with S. Typhimurium. Positive control group was non-treated with Oenothera biennis L. extract but infected with S. Typhimurium on day 0. Two groups were fed with 0.1% and 0.5% of Oenothera biennis L. extract in their diet for 3 days before infection with S. Typhimurium, respectively. They were orally challenged at day 0 with S. Typhimurium and kept for 21 days. Their body weights were measured at days 0, 7, 14, and 21.
jpvm-38-1f2.tif
Fig. 3
Daily gains of pigs in 4 groups were determined at day 21. The statistical significance was indicated at p<0.05 (*).
jpvm-38-1f3.tif
Fig. 4
Feed conversion ratio of pigs in 4 groups was determined at day 21.
jpvm-38-1f4.tif
Fig. 5
Feed efficiency of pigs in 4 groups was determined at day 21.
jpvm-38-1f5.tif
Table 1
Minimum inhibitory concentration (MIC) of Oenothera biennis L. extract against Salmonella spp.
Serotype Origin MIC (mg/mL)
S. Choleraesuis Pig < 0.16
S. Enterica Pig < 0.16
S. Typhimurium Pig < 0.16
S. Enteritidis Chicken < 0.16
S. Gallinarum Chicken < 0.16
Table 2
Scores of pig feces
Groupa Before challenge Day post challenge with S. Typhimurium
1 day 2 day 3 day 7 day 14 day 21 day
1 1 1 1 1 1 1 1
2 1 3 3 3 3 2 1
3 1 3 3 3 2 1 1
4 1 3 3 3 2 1 1

a 1; Pigs in negative control group were neither treated with Oenothera biennis L. extract nor infected with S. Typhimurium. 2; Pigs in positive control group were infected with S. Typhimurium but not treated with Oenothera biennis L. extract. 3; Pigs in this group were fed for 3 days with 0.1% extracts of Oenothera biennis L. extract in their diet and challenged with S. Typhimurium. 4; Pigs in this groups were fed for 3 days with 0.5% extracts of Oenothera biennis L. extract in their diet and challenged with S. Typhimurium. Pigs in groups 3 and 4 were fed for 21 days with 0.1% and 0.5% extracts of Oenothera biennis L. in their diet with after challenge with S. Typhimurium, respectively. Fecal scores: 1 = firm stool, 2 = semi - solid feces, 3 = watery diarrhea, 4 = blood-tinged stool, 5 = profuse bloody diarrhea.

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