Introduction
Probiotics are defined as microorganisms that can provide a beneficial effect on health when ingested in an appropriate amount. These effects include helping in achieving balance of the normal intestinal microflora, inhibiting the formation of harmful bacteria, preventing various diseases and anticancer activity, enhancing the immune system and antioxidant activity, and lowering blood cholesterol (Homma, 1988). Lactobacillus spp. are one of the oldest probiotics and are generally used for farm animals these days (Silva et al., 1999; Jeong et al., 2017). Some previous studies reported that Lactobacillus plantarum has inhibitory ability against pathogenic bacteria. Yang et al. (2014) reported that feeding L. plantarum to young male piglets improved their growth performance by preventing diarrhea induced by E. coli K88 challenge and enhancing the function of the intestinal barrier. Fayol-Messaoudi et al. (2007) reported that intragastric administration of 200 µL of L. plantarum to Salmonella Shigella Typhimurium-infected conventional mice reduced the S. Typhimurium counts in intestinal tissues and intestinal contents. Murry et al. (2004) reported that L. plantarum inhibited the growth of E. coli and S. Typhimurium on normal starter and grower diets for intestinal tract of broiler chickens by reducing the pH level. Many probiotics are lactic acid bacteria and Lactobacillaceae are known for producing various bacteriocins and secondary metabolites with antibacterial activity (Skyttä et al., 1993; Asahara et al., 2004). Pediococcus is a group of lactic acid bacteria and belongs to the family Lactobacillaceae. Pediococcus pentosaceus strains can survive while passing through the digestive tract because they are capable of surviving in the stomach and are resistant to low pH, bile salt, and pepsin (Chiu et al., 2008; Osmanagaoglu et al., 2010; Xu et al., 2018; Park and Choi, 2021). P. pentosaceus strains showed intense inhibitory ability against E. coli and Salmonella Shigella (Silva et al., 2017). This ability might help the P. pentosaceus strains to reach the intestines safely and act as probiotics against pathogenic bacteria. There are many previous studies about the use of probiotic strains in infected animals to investigate their inhibitory ability against pathogenic bacteria; however, there are only a few studies about P. pentosaceus strains. This study was conducted to investigate the protective ability of P. pentosaceus strains, isolated from three different types of Kimchi, against E. coli and S. Typhimurium in E. coli-infected imprinting control region (ICR) mice and S. Typhimurium-infected ICR mice.
Materials and Methods
The experimental protocol for this study was reviewed and approved by the Institutional Animal Care and Use Committee of Chungbuk National University, Cheongju, Korea (approval #CBNUA-1427-20-02).
Microorganisms
E. coli and S. Typhimurium were received from Dankook University (Cheonan, Korea). L. plantarum was isolated from commercial probiotics supplement (Lactoplan, Genebiotech, Gongju, Korea). P. pentosaceus strain A was isolated from Cabbage Kimchi. P. pentosaceus strain B was isolated from Yeolmu Kimchi. P. pentosaceus strain C was isolated from Baek Kimchi. All of P. pentosaceus were received from Sookmyung Women’s University (Seoul, Korea).
Animals and diets
One hundred twenty five weeks old male ICR mice, were purchased from Daehan BioLink (Eumseong, Korea), were housed under a 12 h light/12 h dark cycle in plastic cages with a temperature of 23 ± 3℃. Mice were fed with same diet (Table 1). Mice were divided into six groups based on the initial weight of mice (26 ± 2 g) on each E. coli infected group and S. Typhimurium infected group. Six groups were Normal control group without E. coli and S. Typhimurium orally administrated (NC-; n = 20), Normal control group with E. coli or S. Typhimurium orally administrated (NC+; n = 20), L. plantarum orally administrated group after E. coli or S. Typhimurium orally administrated (LP; n = 20), P. pentosaceus strain A orally administrated group after E. coli or S. Typhimurium orally administrated (PSA; n = 20), P. pentosaceus strain B orally administrated group after E. coli or S. Typhimurium orally administrated (PSB; n = 20), P. pentosaceus strain C orally administrated group after E. coli or S. Typhimurium orally administrated (PSC; n = 20) on each E. coli infected groups and S. Typhimurium infected groups. They were fed standard mouse chow and saline for drinks. They were allowed free access to feed and drinks. All feed, bedding and water dispenser were sterilized.
Experimental design for growth performance
After 3 days of the acclimation period, 200 µL of E. coli (1.0 × 109 CFU·mL-1) or S. Typhimurium (1.0 × 109 CFU·mL-1) were orally administrated to NC+, LP, PSA, PSB and PSC on day 0 to make E. coli infected ICR mice groups and S. Typhimurium infected ICR mice groups. Same amount of PBS was administrated to NC-. 200 µL (1.0 × 108 CFU·mL-1) of microorganisms were orally administrated to LP, PSA, PSB and PSC once every two days for 2 weeks while the same amount of PBS was administrated to NC- and NC+ groups. Body weight (BW) was recorded on day 0, 1, 7, and 14. BW was an important indicator of mice's health status during treatment administration. Body weight gain (BWG), feed intake (FI) and feed efficiency ratio (FER) were measured. All mice were monitored at least once a day to check the mortality rate.
Experimental design for change in the number of microorganisms
To confirm the change of E. coli counts or S. Typhimurium counts in the small intestine, two mouse in each group were dissected on day 0 (administrating E. coli and S. Typhimurium), day 1 and day 7 on each E. coli infected ICR mice groups and S. Typhimurium infected ICR mice groups. Four of mice were dissected on day 14. Intestine contents were isolated from ileum. Feces were collected on day 0, 1, 7, and 14 to confirm the change of E. coli counts or S. Typhimurium counts in large intestine. Intestine contents and feces were diluted in PBS and spread on Macconkey agar (MB cell, Seoul, Korea) or Salmonella Shigella Shigella agar (MB cell, Seoul, Korea) to measure CFU·mL-1 of E. coli or S. Typhimurium. Macconkey agar and Salmonella Shigella Shigella agar were incubated at the incubator for 24 hours and 48 hours, respectively.
Statistical analysis
SAS program (Statistical Analysis System 9.1, SAS Institute, Cary, NC, USA) was used to execute all statistical analyses. One-way ANOVA (SAS Institute, Cary, NC, USA) was used to analyze results statistically. Probability values (p < 0.05) were used to show statistical significance. Tukey’s test was operated for comparison (p < 0.05) when variance was noticed among groups.
Results
Growth performance
Table 2 shows BW, BWG, FI and FER differences according to different treatments in E. coli infected mice group. On day 0 and day 1, there were no significant differences (p > 0.05) among groups in BW. On day 7 and day 14, NC- was the highest (p < 0.05) in BW. LP, PSA, PSB and PSC were significantly higher (p < 0.05) than NC+ while PSC had no significant difference (p > 0.05) with LP.
On week 1 and week 2, NC- was the highest (p < 0.05) in BWG. LP, PSA, PSB and PSC were significantly higher (p < 0.05) than NC+ while PSC had no significant difference (p > 0.05) with LP in BWG. NC- was the highest (p < 0.05) in FI. LP, PSA, PSB and PSC were significantly higher (p < 0.05) than NC+ while there were no significant differences among NC+, LP, PSA, PSB and PSC in FI. NC- was the highest (p< 0.05) in FER. There were no significant differences (p > 0.05) displayed among NC+, LP, PSA, PSB and PSC in FER. Two and one mice have died in NC+ and PSA, respectively.
Table 3 shows BW, BWG, FI and FER differences according to different treatments in S. Typhimurium infected mice group. On day 0 and day 1, there were no significant differences (p > 0.05) among groups in BW. On day 7 and day 14, NC- was the highest (p < 0.05) in BW. LP, PSA, PSB and PSC were significantly higher (p < 0.05) than NC+ while there were no significant differences (p > 0.05) among LP, PSA, PSB and PSC.
On week 1 and week 2, NC- was the highest (p < 0.05) in BWG. LP, PSA, PSB and PSC were significantly higher (p < 0.05) than NC+ while there were no significant differences (p > 0.05) among LP, PSA, PSB and PSC in BWG. NC- was the highest (p < 0.05) in FI. LP, PSA, PSB and PSC were significantly higher (p < 0.05) than NC+ while there were no significant differences (p > 0.05) among LP, PSA, PSB and PSC in FI. NC- was the highest (p < 0.05) in FER. PSA, PSB and PSC were significantly higher (p < 0.05) than NC+ in FER. One of mice has died in NC+.
Microorganism counts change in intestines
Table 4 shows the change of E. coli in the large intestine and small intestine by different treatments. No significant differences (p > 0.01) were shown in the small intestine on day 0 and day 1. NC+ showed the highest (p > 0.01) counts on day 7 while NC- was the lowest (p >0.01). PSC had no significant difference (p>0.01) with LP on day 7. NC+ showed the highest (p > 0.01) counts on day 14 while NC-, LP and PSC were the lowest (p >0.01). No significant differences (p > 0.01) were shown in the large intestine on day 0 and day 1. NC+ showed the highest (p > 0.01) counts on day 7 and day 14 while NC- was the lowest (p> 0.01). PSC had no significant difference (p > 0.01) with LP on day 7 and day 14.
Table 5 shows the change of S. Typhimurium in the large intestine and the small intestine by different treatments. No significant differences (p > 0.01) were shown in the small intestine on day 0 and day 1. NC+ showed the highest (p > 0.01) counts on day 7 while NC- was the lowest (p > 0.01). PSC had no significant difference (p > 0.01) with LP on day 7. NC+ showed the highest (p > 0.01) counts on day 14 while LP and PSC were the lowest (p>0.01). PSC and LP were shown lower (p > 0.01) counts than NC- in the small intestine. No significant differences (p > 0.01) were shown in the large intestine on day 0 and day 1. NC+ showed the highest (p >0.01) counts on day 7 while NC- was the lowest (p > 0.01). PSC had no significant difference (p > 0.01) with LP on day 7. NC+ showed the highest (p >0.01) counts on day 14 while LP was the lowest (p > 0.01). PSC and PSA had no significant difference (p > 0.01) with NC- on day 14.
Discussion
In recent years, E. coli O157:H7 and Salmonella Shigella are the most common pathogens, which cause diarrhea and enteritis (Sung and Ho, 2003; Silva et al., 1999). E. coli O157:H7 is known as Shiga toxin-producing E. coli, which can cause infections even with a small amount (Lee et al., 2017). Salmonella Shigella can cause acute gastroenteritis (Sung and Ho, 2003). In this context, E. coli and Salmonella Shigella can cause reduction in BW, average daily gain (ADG), and FI by infections. Supplementing probiotics may balance the imbalanced intestinal microflora and improve the health performance (Giang et al., 2012). The main purpose of this research was to investigate the protective function of P. pentosaceus strains, isolated from Cabbage, Yeolmu, and Baek Kimchi, against E. coli and S. Typhimurium in E. coli-infected ICR mice and S. Typhimurium-infected ICR mice.
Growth performance
Research about an increase in growth performance by oral administration of P. pentosaceus has been reported in previous studies. Oral administration of 200 µL (3 × 109 CFU·mL-1) of P. pentosaceus LI05 in Clostridium difficile-infected mice reduced weight loss than that in the normal control group (Xu et al., 2018). Oral administration of 1 ml (3 × 109 CFU·mL-1) of P. pentosaceus LI05 in a liver cirrhosis model reduced weight loss compared to that in the normal control group by protecting the intestinal barrier in rats (Shi et al., 2017).
In E. coli-infected ICR mice groups, growth performance was significantly improved in PSA, PSB, and PSC than NC+ by increasing the BW, BWG, and FI. No significant differences were found in FER among groups. PSC showed no significant difference than LP in BW, BWG, FI, and FER. These results are supported by earlier studies. L. plantarum CJLP243 (T3) inhibited E. coli and improved average daily feed intake (ADFI), ADG, and FER (Lee et al., 2012). L. Plantarum B1 can inhibit E. coli K88 and promote growth performance (Wang et al., 2017). Feeding P. acidilactici (1 × 106 CFU·g-1) and P. pentosaceus (1.3 × 106 CFU·g-1) to lambs improved the BWG and FI (Saleem et al., 2017).
In S. Typhimurium-infected ICR mice groups, growth performance was significantly improved in PSA, PSB, and PSC than NC+ by increasing the BW, BWG, and FI. PSA, PSB, and PSC showed no significant difference than LP in BW, BWG, and FI. PSA, PSB, PSC, and LP were higher than NC+ at week 1 in terms of FER. PSA, PSB, and PSC were higher than NC+ and LP at week 2 in terms of FER. These results are supported by a previous study. Feeding L. plantarum CWBI-B659 with xylanase Belfeed B1100MP significantly increased the growth rate in S. Typhimurium-infected broilers (Vandeplas et al., 2009). The piglets fed lactic acid bateria (LAB) complex inclusive of P. pentosaceus D7 showed improved ADG and ADFI and lower feed conversion ratio (FCR) (Giang et al., 2012). Feeding Pediococcus spp to growing lambs through drinking water improved the BW, BWG, and FER (El-Katcha et al., 2016). Feeding 2 × 107 CFU·g-1 of P. pentosaceus with basal diet to SPF chickens improved the BW and ADG and lowered the FCR (Chen et al., 2017).
In both E. coli-infected ICR mice groups and S. Typhimurium-infected ICR mice groups PSA, PSB, and PSC showed higher growth performance than NC+.
P. pentosaceus showed a growth performance improvement effect by inhibiting the growth of E.coli and S. Typhimurium. These results are supported by previous studies. P. pentosaceus can inhibit E. coli and S. Typhimurium. P. pentosaceus MP12, P. pentosaceus MP-22, P. pentosaceus MP-36 isolated from pickled cabbage have been confirmed to have strong inhibitory activity against E. coli and S. Typhimurium (Chiu et al., 2008). P. pentosaceus M7-1 and P. pentosaceus DPC6006 have strong inhibitory ability against E. coli O157:H7 and S. Typhimurium (Casey et al., 2004).
In E. coli-infected ICR mice groups, two and one mice have died in NC and PSA, respectively. We consider that this occurred due to the low inhibitory ability of P. pentosaceus strain A against E. coli. The experimental results may vary depending on the amount of treatment and the number of samples. In S. Typhimurium- infected ICR mice groups, none of the mice have died in PSA, PSB, and PSC. It was a different result from the previous study. Pretreatment with P. pentosaceus 40 accelerated the infection and increased the number of deaths (Silva et al., 2017). The difference in experimental results depend on the pretreatment or not, number of test samples, environment, and diet composition.
Microorganism count change in intestines
Previous studies reported that weaned piglets fed LAB have lower E. coli count in the intestine (Tortuero et al., 1995; Huang et al., 2004; Mallo et al., 2010). Some strains of P. pentosaceus have shown outstanding antimicrobial activity against pathogenic bacteria, like E. coli and Salmonella Shigella (Xu et al., 2018; Lan et al., 2020). In this context, P. pentosaceus can reduce pathogenic bacterial counts in the intestine. A higher number of E. coli and S. Typhimurium were significantly reduced in PSA, PSB, and PSC than NC+ in the small intestine and the large intestine on day 7 and day 14. In terms of reduction of E. coli, PSC showed no significant difference than LP in the small intestine and the large intestine on day 7 and day 14, while it showed no significant difference than NC- in the small intestine on day 14. In the reduction of S. Typhimurium, PSC showed no significant difference than LP in the small intestine (on day 7 and day 14) and the large intestine (on day 7), while it showed no significant difference in NC- treatment in the large intestine on day 14. A higher number of S. Typhimurium was significantly reduced in PSC than NC- in the small intestine on day 14. This result is consistent with previous studies. L. Plantarum B1 reduced the E. coli count in the cecal content (Wang et al., 2017). L. Plantarum ZS2058 can inhibit Salmonella Shigella in a murine model (Liu et al., 2019). Feeding 2 × 107 CFU·g-1 of P. pentosaceus basal diet to SPF chickens decreased the number of E. coli in the cecal content. (Chen et al., 2017). P. pentosaceus MP12, P. pentosaceus MP-22, and P. pentosaceus MP-36 isolated from pickled cabbage significantly reduced the Salmonella Shigella count in the spleen and liver of mice (Chiu et al., 2008). We are in agreement with the results of (Lan et al., 2020), which suggested that the reduction in E. coli and S. Typhimurium in the intestines is due to P. pentosaceus expelled organic acid to lower the pH value and produce secondary metabolites, like a bacteriostatic toxin.
Conclusion
In conclusion, we considered that P. pentosaceus strains, isolated from three different types of Kimchi, can act as probiotics by inhibiting E. coli and S. Typhimurium. Among these three P. pentosaceus strains, P. pentosaceus strain C showed the highest inhibitory ability against E. coli and S. Typhimurium due to the highest growth performance and reduced the maximum number of E. coli and S. Typhimurium in the intestines; however, it showed no significant difference than L. plantarum (except for S. Typhimurium counts in the large intestine on day 14).
Acknowledgments
This work was supported by a grant from the Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea [Z-1543081-2020-22-01].
Authors Information
Han Jin Oh, http://orcid.org/0000-0002-3396-483X
Jun Pyo Lee, http://orcid.org/0000-0001-7224-3958
Ji Hwan Lee, http://orcid.org/0000-0001-8161-4853
Yong Ju Kim, http://orcid.org/0000-0002-0960-0884
Jae Woo An, http://orcid.org/0000-0002-5602-5499
Se Yeon Chang, http://orcid.org/0000-0002-5238-2982
Young Bin Go, http://orcid.org/0000-0002-5351-6970
Dong Cheol Song, http://orcid.org/0000-0002-5704-603X
Hyun Ah Cho, http://orcid.org/0000-0003-3469-6715
Min Gyu Jeon, http://orcid.org/0000-0003-2003-1130
Yo Han Yoon, http://orcid.org/0000-0002-4561-6218
Jin Ho Cho, http://orcid.org/0000-0001-7151-0778
