Probiotic can Prevent Sepsis in Rats Induced by Lipopolysaccharide Escherichia coli

 

Efris Kartika Sari*, Titin Andri Wihastuti, Priska Rizqi Afenia, Wahyu Ardiansyah

School of Nursing, Faculty of Medicine, University of Brawijaya, Malang, Indonesia.

*Corresponding Author E-mail: efriskartika@ub.ac.id

 

ABSTRACT:

Escherichia coli bacterial infection (E. coli) is the cause of sepsis in critically ill patients. Consumption of probiotic is one attempt to increase endurance against bacterial infections. This study aimed to determine the effect of probiotic Lactobacillus spp. on pro-inflammatory cytokines (interleukin-1 (IL-1)), sepsis markers (procalcitonin (PCT) and white blood cell (WBC) count in rats (Rattus norvegicus) induced by lipopolysaccharide (LPS) E. coli. Twenty-one rats were divided into three groups: (1) group of healthy control rats, (2) group of rats induced by LPS E. coli, and (3) group of rats treated by Lactobacillus spp. and induced by LPS E. coli. The group of rats treated by Lactobacillus spp. and induced by LPS E. coli was treated with additional Lactobacillus spp. probiotic on day 1-14, then induced with LPS E. coli on day 15. All blood samples were taken on the 16th day. IL-1 and PCT levels were analyzed with the ELISA method, the WBC count was measured with improved Neubauer hemocytometer. The group of rats induced by LPS E. coli  had the highest IL-1, PCT, and WBC count. The statistical analysis of IL-1 did not show a significant difference (p > 0.05), but PCT and WBC count showed significant difference when compared with group of rats treated by Lactobacillus spp. (p < 0.05). Probiotic Lactobacillus spp. can prevent sepsis in rats induced by LPS E. coli.

 

KEYWORDS: Lactobacillus spp., Lipopolysaccharide E. coli, IL-1, PCT, WBC count.

 

 


INTRODUCTION:

Nosocomial infection is one of the biggest causes of sepsis in critically ill patients admitted to intensive care unit. About 75% of the burden of nosocomial infections is present in developing countries. Most of these infections are caused by gram-negative bacteria originating from the gastrointestinal tract such as Escherichia coli (E. coli)1-3. Infections that develop into sepsis have been shown to increase the length of stay, cost of care, morbidity, and patient mortality4.

 

An essential product in the occurrence of sepsis is lipopolysaccharide (LPS) which is the outer membrane component of gram-negative bacteria. LPS E. coli is a glycolipid complex, the main component of the outer wall of gram negative-bacteria with an endotoxin characteristic and capable of stimulating immune cells5, 6.

 

Induction of LPS E. coli in rats causes LPS to bind to specific proteins in the plasma through Lipopolysaccharide Binding Protein (LPB). Furthermore, LPS and LPB will integrate with Cluster Differentiation 14 (CD14) and Toll-Like Receptor 4 (TLR 4) to activate protein regulation (Nuclear Factor kappa Beta/NFkB). Activated cells secrete pro-inflammatory mediators, namely cytokines (TNF-α, IL-1, IL-6), chemokine (IL-8), prostaglandin and histamine. The mediator works on blood vessel endothelial cells, causing an inflammatory response, namely vasodilation, increased vascular permeability, and recruitment of neutrophils to tissues7, 8.

 

The infection can develop into sepsis if the infectious agent secretes the toxin continuously. As a result, an inflammatory response persists through continuous activation of inflammatory mediators, cellular hypoxia, tissue damage, shock, and organ failure, and has the potential to cause death9. The detection of the infected condition and its development into sepsis can be seen from several parameters, namely levels interleukin-1 (IL-1), pro-calcitonin (PCT), and white blood cell (WBC) count10-13.

The body responds to infection by secreting pro-inflammatory cytokines, including IL-1. IL-1 has a significant role in mediating innate and adaptive immune responses to various infectious diseases14. Studies by Pierrakos and Vincent show that pro-inflammatory cytokines, especially IL-1, is known to have increased in sepsis patients compared to non-sepsis patients15. The body also responds to infection by increasing WBC count which explains why WBC counts are used to indicate the presence of infection and to follow the development of infection into sepsis, which is when leukocytosis occurs (WBC count > 12,000 µL -1) or when leukopenia occurs (WBC count < 4,000 µL -1).11 PCT is used to determine whether the condition of the infection has developed into sepsis. To mark sepsis in the acute phase, PCT is used due to its sensitivity and specificity in determining the prognosis of sepsis. Clinical trials have shown that PCT is a specific sign of severe bacterial infections, and can distinguish between patients with sepsis and patients with response systemic inflammatory syndrome (SIRS)15, 16.

 

The inflammatory response is a body mechanism that aims to increase the immune response to eliminate pathogenic microorganisms. If the body's inflammatory response is adequate, the infection can be controlled and resolved. However, if the body cannot properly regulate the secretion of the mediator, sepsis will occur. One preventive effort to ensure that the infection condition does not occur and develop into sepsis is by increasing the body's immunity through the administration of lactic acid bacteria (BAL) which is isolated into probiotic17, 18.

 

Lactobacillus spp. bacteria that are included in BAL have been widely consumed for its health benefits19. Lactobacillus spp. are able to “balance” unfriendly bacteria because when they produce lactic acid, they alter the intestinal environment, making it unsuitable for unfriendly bacteria20. Most research on the effect of probiotics focuses on preventing and treating gastrointestinal disease, even though probiotics have the potential to prevent infection from becoming sepsis. This study aims to determine the effect of probiotic Lactobacillus spp.  on pro-inflammatory cytokines IL-1, and sepsis markers (PCT and WBC count) in rats  induced by LPS E. coli.

 

MATERIALS AND METHODS:

This study was an experimental study. This was conducted in Microbiology Laboratory and Parasitology Laboratory, Faculty of Medicine, University of Brawijaya from December 2016 to February 2017.

 

Culture Preparation of Lactobacillus spp.:

Lactobacillus spp. was obtained from the Microbiology Laboratory, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia. The preparation procedure for the probiotic cultures of Lactobacillus spp. began by sterilizing a curved inoculating loop by heating the loop above the bunsen burner until it was red hot. After the loop had cooled, the Lactobacillus spp. was taken from the stock by touching the end of the loop to the stock, then it was scratched onto the surface of the media MRS-Agar with a streaking quadrant method to obtain separate colonies. It was followed by incubating the media for 37o C for 24 hours. The grown pure colony was re-identified to determine the bacterial species. After the colony was proven as Lactobacillus spp. species, a culture enrichment was done to produce a large amount, and dilution was done to reach the desired bacterial density21. Lactobacillus spp was administered a 0.5 ml dose of 109 CFU/Kg BW/day dissolved in the media dextrose 5% (D5%). Probiotic was administered through the gastric tube once a day for 14 days to achieve the optimal effect4, 22.

 

Rats:

Rats (Rattus norvegicus) of Wistar strain, male, aged 10 - 12 weeks, weighed at 150 - 200 grams were obtained from the Faculty of Medicine, University of Brawijaya. Before being treated, the animals were acclimatized for seven days by being fed through an ad-libitum method. The use of experimental animals as a subject in this research has passed the ethical conduct from the Ethics Commission of Health Research, Faculty of Medicine, University of Brawijaya.

 

Induction of LPS E. coli:

Induction of LPS E. coli (Biological List, catalog number 201) was done orally. LPS was diluted with NaCl 0,9%   solution, with a ratio of NaCl 0.9% : LPS E. coli  of 10 : 1. The dose given was 1 mg/Kg BW dissolved in 0.5 ml NaCl 0.9% media23.

 

Research Design:

Twenty-one rats were randomly divided into 3 groups as follows: group of healthy control rats (healthy group, n= 7); group of rats induced by LPS E. coli (LPS group, n= 7) with a dose of 1 mg/Kg BW on day 15; and group of rats treated by probiotic Lactobacillus spp. (109 CFU/Kg BW/day for 14 days), and induced by LPS E. coli (LPS + LB group, n= 7) at a dose of 1 mg/Kg BW on the 15th day. Intra-cardiac blood sampling was performed on day 16 (24 hours following induction of E. coli LPS).

 

Measurement of IL-1 and PCT levels:

The measurement of IL-1 and PCT levels was analyzed using enzyme-linked immunosorbent assay (ELISA) kit namely RayBio® Rat IL-1 beta ELISA Kit no. ELR-IL1b catalog, and MyBioSource no. catalog of MBS760081. The sensitivity of the IL-1 and PCT kits were 80 pg/ml and < 9.375 pg/ml.

 

Measurement of WBC Count:

The measurement of WBC count was calculated using counting chambers (improved Neubauer). The calculation of white blood cell count is carried out in four areas of counted chambers. The number of WBC per mm3 of blood is obtained as follows: number of WBCs counted x blood dilution factor x chamber depth divided by the area of ​​chamber counted= source of WBCs counted x 50 (Blood dilution factor= 20; Chamber depth= 10; Area of ​​chamber counted= 4)24.

 

Statistical Analysis:

Data were presented as mean value ± standard deviation (SD). Statistical tests were performed using SPSS statistical software (version 2.0, Chicago, IL, USA). One way ANOVA test was done to determine the effect of probiotic on the levels of IL-1, PCT, and WBC count in each group. Comparison of the effect of probiotic between groups was analyzed by the Post hoc test (Tukey's test).

 

RESULTS:

Effect of Probiotic on IL-1 Levels:

The difference of IL-1 levels among study groups was demonstrated in Table-1.

 

Table-1 The Difference of IL-1 levels in the group of healthy control rats, group of rats induced by LPS E. coli, and group of rats treated by Lactobacillus spp. and induced by LPS E. coli

Study Group

IL-1 Levels (pg/ml)

P-Value

Mean ± SD

Range

Median (IQR)

Healthy group

17.78 ± 2,64

(14.59-23.05)

17.57 (2.06)

0.952

LPS group

18.00 ± 5,56

(11.86-29.17)

16.55 (5.10)

LPS + LB group

17.36 ± 2,91

(14.59-23.05)

16.55 (4.02)

The highest level of IL-1 was found in the group of rats induced by LPS E. coli. However, there was no significant difference in IL-1 levels among healthy group, LPS group, and LPS + LB group (p > 0.05).

 

Effect of Probiotic on PCT Levels:

The difference of PCT levels among study groups was demonstrated in Table-2.

 

Table-2 The Difference of PCT levels in the group of healthy control rats, group of rats induced by LPS E. coli, and group of rats treated by Lactobacillus spp. and induced by LPS E. coli

Study Group

PCT Levels (pg/ml)

P-Value

Mean ± SD

Range

Median (IQR)

Healthy group

13.73 ± 0,14

(13.59-13.97)

13.67 (0.24)

0.000

LPS group

14.61 ± 0,38

(14.18-15.22)

14.51 (0.68)

LPS + LB group

13.88 ± 0,23

(13.52-14.16)

13.91 (0.44)

 

The highest PCT level was found in the group of rats induced by LPS E. coli. PCT level in the LPS group increased significantly when compared to the healthy group (14.61 ± 0.38 pg/mL vs. 13.73 ± 0.14 pg/mL; p < 0.001) (Figure-1). PCT level on the LPS group also increased significantly when compared with the LPS + LB group (14.61 ± 0.38 pg/mL vs. 13.88 ± 0.23 pg/mL; p < 0.001). The difference in PCT levels in the healthy group with the LP + LB group was not statistically significant (13.73 ± 0.14 pg/mL vs 13.88 ± 0.23 pg/mL, p > 0.05).

 

Figure 1. Comparison of PCT levels in group of healthy control rats, group of rats induced by LPS E. coli, and group of rats treated by Lactobacillus spp. and induced by LPS E. coli.

 

Effect of Probiotic on WBC Count:

The difference of WBC count among study groups was demonstrated in Table-3.

 

Table-2 The Difference of WBC counts in the group of healthy control rats, group of rats induced by LPS E. coli, and group of rats treated by Lactobacillus spp. and induced by LPS E. coli

Study Group

WBC Counts (cell/µl)

P-Value

Mean ± SD

Range

Median (IQR)

Healthy group

6,992.86 ± 1,110.34

(5,700-8,400)

6,800 (2,250)

0.000

LPS group

29,478,57 ± 3064,02

(25,500-34,650)

30,000 (4,200)

LPS + LB group

26,535.71 ± 1,773.58

(24,000-29,000)

26,000 (3,150)

 

The WBC count in both groups of rats induced by LPS E. coli showed an increase, with the highest number found in the group of rats which was only induced by LPS E. coli without treated by probiotic. The WBC count in the LPS group increased significantly when compared with the healthy group (29478.57 ± 3064.02 cell/µL vs. 6992.86 ± 1110.34 cell/µL; p < 0.001) (Figure-2). The WBC count in the LPS + LB group also significantly increased when compared with the healthy group (26535.71 ± 1773.58 cell / µL vs. 6992.86 ± 1110.34 cell/µL, p < 0.001). Although they both experienced an increase in WBC counts, the difference in the WBC counts in the LPS group with the LPS + LB group showed statistically significant difference (29478.57 ± 3064.02 cell/µL vs. 26535.71 ± 1773.58 cell/µL, p < 0.05).

 

Figure-2. Comparison of WBC counts in the healthy control group of rats, the group of rats induced by LPS E. coli, and the group of rats treated by Lactobacillus spp. and induced by LPS E. coli.

 

DISCUSSION:

IL-1, PCT, and the number of WBC play an essential role in the inflammatory process and are widely used to identify the occurrence of sepsis. Sepsis induction can use an LPS E. coli dosage of 0.1 mg/Kg BW - 40 mg/Kg BW. In the use of low doses, the effect of sepsis persists for up to 48 hours. An oral dose of 1 mg/Kg BW will result in a maximum immune response within 24 hours after exposure23, 25. The pathological characteristics of sepsis is the inability of the body to maintain a balance of pro-inflammatory and anti-inflammatory mediators resulting in a systemic inflammatory response26. The results of this research show that the highest levels of IL-1, PCT, and WBC counts were found in the group of rats induced by LPS E. coli, compared to the group of healthy control rats, and the group of rats treated by Lactobacillus spp. and induced by LPS E. coli.

 

Induction of LPS E. coli in rats causes resident immune cells such as macrophages generate a pro-inflammatory state in response to pathogen-associated molecular patterns (PAMPs) from the LPS E. coli, and damage-associated molecular patterns (DAMPs) that are released by rats cells. Resident immune cells recognize these patterns mainly via Toll-like and lectin receptors and then release inflammatory mediators to attract other immune cells such as neutrophils and cells of adaptive immunity to the site of infection in order to eliminate the pathogen without harming the host. First responding cells are monocytes and macrophages, which through induction of early response genes such as TNF-α induce inflammatory cytokines and chemokines, amplifying the inflammatory response. This is followed by activation of lymphocytes as an adaptive immune response, as well as the coordination of later phases of the immune response. Additionally, nitric oxide (NO) that is produced by endothelial cells induces vasodilation and an increase in WBC delivery to the sites of immune-activity6, 27.

 

IL-1 is one of the major resulting pro-inflammatory cytokines if the body experiences an infection in the acute phase, and also experience an increase in sepsis patients compared to patients of non-sepsis. In this study, blood plasma sampling for measurement of IL-1 levels was carried out 24 hours after the induction of LPS E. coli. The three groups showed almost the same value. This is probably due to a temporary increase in pro-inflammatory cytokine levels, such as IL-1, which reaches a peak of 1.5-4.5 hours after bacterial induction, and after that gradually returns to normal23. This is in accordance with the results of the McAllister et al. study, which reported the results of cytokine measurements in three patients with sepsis conditions after receiving erythrocyte transfusion (PRC) contaminated with gram negative bacteria. IL-1 in the two surviving patients were detected to reach the peak at four hours after exposure, then gradually returned to normal. Whereas in one patient who later died, IL-1 levels remained high for 22 hours28.

 

WBC count in both LPS group and LPS + LPB group that received LPS E. coli induction both increased. This is caused by an inflammatory response to the induction of LPS E. coli in the body of white rats for 24 hours. This is consistent with research conducted by Penailillo et al. which showed that intravenous administration of LPS E. coli in rabbits caused a significant increase in total WBC and neutrophils count in samples taken at 12 and 24 h after the first injection of LPS29. E. coli infection will affect the hematologic system, one of which is the number of white blood cells as the body's first line of defense against infection and inflammatory conditions.

 

In most patients, the pro-inflammatory response is self-limiting, but in some patients the response becomes exaggerated, which leads to sepsis. PCT is a prohormone calcitonin produced by the liver, kidney, adipose, and parenchymal muscle cells in response to bacterial endotoxin, and is widely used as a biomarker of sepsis30. Numerous studies have investigated the diagnostic usefulness of PCT, comparing it with CRP. Initially, PCT was found more sensitive and specific than CRP for bacterial infection. PCT levels play a role in detecting sepsis while predicting the patient's prognosis. A study by Nanda et al. showed that patients with high PCT levels were more at risk of death when compared with patients with low PCT levels31.

 

The results of this study also showed that PCT levels and WBC counts increased significantly in the LPS group, compared to the healthy group, and the LPS + LB group. However, there were no significant differences in PCT levels in the healthy group with the LPS + LB group.  In addition, even though both experienced an increase, the WBC count in the LPS group increased significantly when compared with the LPS + LB group. This shows that a worse infection occurred in the group of rats that were only induced by LPS E. coli. The Lactobacillus spp. probiotic positively influenced it and stopped the infection to become more severe/sepsis. When Lactobacillus spp probiotic were given to the rats for 14 days as an immunomodulator, it can suppress the inflammatory response and prevent it from going into a sepsis condition. The inflammatory response that occurred in the group of rats treated by probiotics and induction of LPS E. coli will be local and can be defended by the cellular immune system that has been strengthened by probiotic32.

 

Some postulate can explain the mechanism of probiotics in preventing the development of infection into sepsis, ie: the ability to obtaining an attachment site, the ability to influence the intestinal pH, improve the function of the intestinal wall, microfloral modification with the synthesis of anti-microbes compounds, triggering immune response, disturbing communication between bacteria, and the ability to compete to obtain nutrition33-37. Probiotic strains of lactobacilli can attach to the epithelium and act as walls against pathogens (colonization barriers) by preventing pathogens from attaching to the gastrointestinal mucosa. Lactic acid and acetic acid produced by probiotics reduce luminal pH which results in a less conducive environment to pathogenic organisms. Probiotic can also improve the complex function of the intestinal walls and can control the stability of cytoskeletal cells through mucus production, chloride, and water secretion. In addition, many types of lactobacilli produce bacteriocins, compounds produced by bacteria with a portion of biologically active proteins and are bactericidal38, 39. Furthermore, probiotics are also known to increase immunoglobulin-A (IgA) secretion, increase the number of natural killer cells, or increase phagocyte macrophage activity. Increased IgA secretion can reduce the number of pathogenic organisms in the intestine thereby increasing the composition of the microflora. The type of lactobacilli is also known to secrete molecules that interfere with communication between bacteria and reduce the expression of virulence of E. coli. probiotic which also use the necessary nutrients for the growth of pathogenic bacteria that can inhibit the growth of pathogen bacteria17, 40, 41.

 

The mechanism of Lactobacillus in inhibiting the action of infection-induced LPS is probiotic not only alleviates inflammation, but also has an immunostimulatory effect on intestinal epithelial cells. Previous study demonstrated that Lactobacillus rhamnosus GG alleviates inflammation in LPS-stimulated porcine intestinal epithelial cells by modulating Toll-like receptors (TLRs) and inhibiting mitogenactivated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) signaling42.

 

The conclusion of this study is; probiotic Lactobacillus spp. can prevent sepsis in rats induced by LPS E. coli. The administration of probiotic Lactobacillus spp. can be beneficial in preventing increased PCT levels and WBC counts in rats induced by LPS E. coli, where PCT levels and WBC counts are markers of sepsis. Further studies are needed to determine the effective dose of probiotic Lactobacillus spp. in preventing sepsis caused by infection of E. coli bacteria.

 

ACKNOWLEDGEMENT: 

The authors are grateful to the Faculty of Medicine, University of Brawijaya that funded the study.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 01.09.2020            Modified on 28.12.2020

Accepted on 08.02.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2021; 14(12):6315-6320.

DOI: 10.52711/0974-360X.2021.01092