Probiotics for the Management of Sepsis: Advances in Animal Models and Intensive Care Unit Environments

Abstract

Sepsis frequently leads to multiple organ failure and is a major cause of morbidity and mortality in critically ill patients. Although intensive care protocols and antibiotic therapy have improved sepsis treatment, specific management is lacking with respect to efficient protection from tissue damage and long-term outcomes. Probiotics are live microbes that modulate the immune system and inflammation and colonize the gut. In this narrative review, we have traced the evolution of the administration of probiotics in an animal model of sepsis and treatment alternatives in the intensive care unit setting. First, probiotics are categorized by species before describing their modulation of the microbiota, repair of tissue-specific damage, immune response, and molecular pathways to prevent complications. The impact on therapy for infant and adult patients is also addressed. Finally, we have emphasized the challenges and gaps in current studies as well as future perspectives for further investigation. The present review can open up avenues for new strategies that employ promising probiotic strains for the treatment of sepsis and discusses their ability to prevent disease-associated long-term complications.

1. Introduction

Sepsis is a life-threatening condition that poses a public health risk. Despite persistent socioeconomic differences throughout the world, levels of morbidity and mortality due to sepsis remain high, imposing a burden on healthcare systems worldwide. Access to and quality of healthcare contribute to lower rates of complications during sepsis. However, underreporting of sepsis cases negatively impacts treatment and impedes the delivery of healthcare services [1,2,3].

Inflammation triggered by host defense mechanisms against infectious agents, when not properly kept in check by immunoregulatory mechanisms, can lead to tissue damage and multiple organ failure [4,5]. Even with vasopressor and intravenous fluid interventions, early and aggressive antibiotic administration has been administered during the hyperinflammatory phase to prevent complications and death due to sepsis [6]. Unfortunately, antibiotic therapy affects the intestinal microbiome by promoting dysbiosis and might contribute to gastrointestinal tract (gut) damage. In this regard, intestinal microbiota can be associated with the long-term progression in the ICU environment, being a trend of clinical research. Microbiota alteration can predict the risk of sepsis and its mortality based on the abundance of certain harmful gut microbes [7]. Antimicrobial resistance is another factor that impedes recovery despite therapy [8]. In addition, the lack of resources to perform interventions in individuals with sepsis might contribute to the high mortality rates observed in middle-income countries [9,10]. Furthermore, sepsis survivors develop post-sepsis syndrome with physical and/or psychological long-term effects, necessitating re-hospitalization due to new bacterial infections [11].

Intestinal epithelial integrity, as well as interactions between the immune system and intestinal microbiota, protect the host and maintain gut homeostasis. However, sepsis disrupts this epithelial barrier by triggering enterocyte apoptosis and reducing cell proliferation, which compromises the local microbiome after immune response activation [12,13,14]. The gut microbiota composition and the microbial metabolite profiles are different in healthy patients than in individuals with sepsis. Patients with sepsis have enteric dysbiosis associated with organ injury [15]. Microbial growth has been associated with inflammation, and loss of microbial diversity can be used to predict the risk of sepsis in an intensive care unit (ICU) environment by evaluating the presence and growth of harmful gut microbes [7]. Although it is known that the gut is involved in sepsis, its exact roles in the progression of organ dysfunction and the therapeutic modulation of dysbiosis are still not clear. Studies have demonstrated that treatment is required to restore mucosal integrity, ameliorate the local immune response, and reverse dysbiosis in critically ill patients, including those with sepsis [12,13,16,17].

In order to promote gut homeostasis by neutralizing the growth of harmful microorganisms and/or reinforcing local immunoregulatory networks, probiotics have been developed as supplements with health benefits. In recent years, the biological effects of probiotics have been explored to increase their spectrum of prophylactic and therapeutic modulation in humans and animals [18,19]. If administered in adequate amounts, these live microorganisms can provide measurable physiological benefits to ailing and immunocompromised individuals [20]. Furthermore, probiotics could play important roles in attenuating sepsis and enterocolitis [21,22].

Based on the potential colonization of probiotics and their ability to prevent tissue dysfunction triggered by inflammatory mediators, it is necessary to clarify the mechanisms involved in the restoration of homeostasis by probiotics during sepsis. In this review, we will describe the effects of probiotic administration on a cecal ligation and puncture (CLP) model, a type of polymicrobial sepsis that mimics the complexity of human disease. Finally, we will emphasize the clinical aspects of probiotics in sepsis management among ICU patients.

Between January 2020 and June 2020, a literature search was conducted on the PubMed database. For an experimental model, the following search terms were used in combination: “CLP”, “polymicrobial sepsis”, “probiotic”, and treatment”. For human studies, we selected previous studies that demonstrated the probiotic therapy in the hospital environment. The following search terms were used in combination: “bacteremia”, “ICU”, “human”, “hospital”, “probiotic”, and “sepsis”. All studies published in the English language were considered, and a date restriction was not applied. In addition, authors reviewed the studies and selected those based on relevance to the topic. Additional articles were identified by manually searching reference lists of included articles.

2. Lactobacillus Rhamnosus GG (LGG)

LGG is a common microbial strain used in basic and applied research. It is characterized as a Gram-positive bacterium that can tolerate different oxygen levels and is found in healthy human intestines. Furthermore, LGG has been consumed as a probiotic supplement for a normal diet and a healthy balance of gut bacteria [23,24]. Many studies have reported the beneficial effects of LGG administration in gastrointestinal [25], allergy [26], and lung [27] diseases. As a result, LGG is a promising agent for modulatory activity in inflammatory disorders, including sepsis.

The prophylactic effect of LGG in mice subjected to sepsis was first addressed by Khailova et al. [28]. The oral administration (p.o.) of LGG immediately before CLP-induced sepsis protects mice from lung damage through the inhibition of neutrophil migration. Importantly, the lung expression of inflammatory markers, such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2, is abrogated. Furthermore, the expression of Toll-like receptor (TLR)-2, its proximal protein myeloid differentiation primary response 88 (MyD88), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) genes, downstream of TLR-2, is inhibited by LGG treatment.

To demonstrate the protective effect of LGG on sepsis, Chen et al. administered LGG (p.o.) daily to mice for 1 month and induced sepsis via CLP. Consequently, the time of death was delayed by several days after probiotic prophylaxis. In addition, LGG attenuated mucosal damage to the ileum, enhanced gut barrier integrity, and normalized lysophosphatidylcholine metabolite levels, which are known to be proinflammatory markers [29]. However, the mechanisms involved in these processes are not known. Based on the same condition of probiotic pre-administration, the group focused on modulation of the gut microbiome, mucosal barrier homeostasis, and inflammatory markers [30]. After colonization, LGG reversed the dysbiosis that was associated with sepsis and improved bacterial diversity, both of which have been proposed as therapeutic targets [31]. Although LGG inhibited the release of IL-2 and IL-22, the levels of TNFα and IL-6 were similar to those in septic mice without probiotic supplementation. Furthermore, Chen et al. [30] reported that LGG prevented epithelial cell apoptosis and improved epithelial tight junctions after the onset of sepsis. Recently, the group demonstrated that LGG was able to reduce bacteremia and restore colon microbiome homeostasis [32].

The impact of LGG administration on polymicrobial sepsis was also evaluated by Ding et al. [33], who determined whether this probiotic could modulate sepsis-related liver injury. Rats that received LGG (p.o.) once immediately prior to insult were protected from death caused by sepsis. The modulatory activities of LGG treatment in alleviating liver damage were associated with inhibition of the expression and/or release of IL-1β, IL-6, NLRP3 inflammasomes, TNFα, vascular endothelial growth factor, and monocyte chemoattractant protein 1, as well as the reduction of oxidative stress and lipid peroxidation. Furthermore, LGG downregulated NF-κB and hypoxia-inducible factor 1-α, indicating an important mechanism by which the probiotic alleviates a cytokine storm.

3. Bifidobacterium Longum

Bifidobacterium longum is another probiotic used in basic and clinical studies. This Gram-positive microorganism has shown positive effects on modulation of the gut [34,35,36], obesity [37], anxiety, stress, depression [38,39,40], and cytokines [41,42]. Khailova et al. [28] demonstrated that the previous administration of B. longum could protect septic mice from lung injury, decreasing TNFα and IL-6 levels, along with levels of COX-2, TLR-2, and MyD88. However, probiotic prophylaxis did not affect TLR-4 and NF-κB expression in the lung tissue.

4. Escherichia Coli

In general, E. coli is a facultative anaerobic Gram-negative bacterium that adapts to diverse environments. Most strains colonize the gut of warm-blooded animals as part of the normal microbiota. Furthermore, E. coli has an extraordinary ability to survive in soil, water, sediment, and food environments [43,44]. Although this bacterium is beneficial to the healthy microbiome, some strains can be extremely virulent in both extraintestinal and intestinal environments. These disorders are caused by the contamination of food products, water, and milk [43,45,46]. However, E. coli has been used by the biotechnology industry as a recombinant therapeutic resource [47]. E. coli Nissle 1917 (EcN) is a probiotic and the most promising strain that exhibits protective activities against different inflammatory disorders, such as allergies [48,49], obesity [50], intestinal inflammation [51,52,53], tumor growth [54,55], and autoimmune dysfunction [56]. Hence, it may be an excellent therapeutic agent for sepsis.

Recently, Guo et al. [57] reported the protective effect of EcN on the intestinal barrier functions of septic mice. An EcN suspension was administered p.o. 2 weeks before the CLP procedure. In the small intestine, the treatment reversed the loss of tight junction proteins, such as zona occludens-1 (ZO-1) and claudin-1, and it slightly downregulated claudin-2. These markers are involved in maintenance of the correct architecture and intestinal barrier permeability. Furthermore, these researchers demonstrated that the supernatant growth medium of EcN was also able to regulate tight junction proteins and inhibit NF-κB and myosin light-chain kinase activation in vitro.

5. Zymomonas Mobilis

Zymomonas mobilis is an anaerobic Gram-negative bacterium used in the metabolic engineering of alcohol production [58,59,60]. This ethanogenic strain produces secondary metabolites with antileukemic properties [61] and is able to abolish Schistosoma mansoni infections [62]. Furthermore, Z. mobilis has been used as a dietary supplement in animal husbandry [63] as it also normalizes intestinal transit and has antilipidemic properties, attenuating cholesterol, and lipoprotein fractions [64].

Campos et al. [65] described the role of Z. mobilis on an experimental model of sepsis. These researchers demonstrated that pretreatment or its association with post-treatment (p.o.) protected mice from severe sepsis. In addition, lung and spleen damage were attenuated due to a decrease in myeloperoxidase levels and apoptotic cells. Bacterial growth and neutrophil migration to local injury were inhibited with the administration of Z. mobilis. The upregulation of IL-10 was found to have a protective role, whereas TNF-α levels, also downmodulated by Z. mobilis, were found to be responsible for organ dysfunction and an increase in the rate of mortality during sepsis.

6. Probiotic Combinations

Several studies have focused on the benefits of probiotic strains and their combinations that can colonize the intestine and modulate immunological responses in gut disorders and inflammatory diseases [66,67,68,69,70,71,72]. Bacillus subtilis and Enterococcus faecium, both Gram-positive microorganisms, have been used as probiotic supplements and regulate microbiota [73,74] and obesity [75,76], have antioxidant and antimicrobial properties [77,78], repair disruptions in the intestinal barrier [79,80], attenuate elevated cholesterol levels [81], and modulate intestinal mucosal immune responses [82]. The oral administration of a mixture of B. subtilis and E. faecium was found to improve the host response of mice challenged with the CLP procedure [83]. A once-daily treatment for 1 week delayed animal death and improved the survival rate. These probiotics upregulate ZO-1, claudin-1, and occludin proteins and attenuated proinflammatory macrophage activation in the ileum. Furthermore, levels of IL-6 and TNF-α were found to be decreased in the serum and ileum tissue. Levels of histamine, a marker associated with mast cell activation, were also attenuated in the serum and the peritoneum. Interestingly, the combination of probiotics did not modulate the levels of anti-inflammatory markers such as TGF-β, IL-10, and type 2 macrophage. The phosphorylation of the serine/threonine-specific protein kinase AKT was also found to be increased with treatment [83].

Biological activities reported previously herein, emphasizing the immunomodulatory and anti-inflammatory activities and the mechanisms of actions of probiotics, are summarized in

Table 1. Experimental designs and immunomodulatory activities of probiotics on cecal ligation and puncture model.

Probiotic	Reference	Probiotic Regime	Immunomodulatory Activities
Bifidobacterium longum	[28]	1 × 107 CFU/mL immediately before CLP	↓ IL6 and TNF-α mRNA expression and levels ↓ COX-2, TLR-2 and MyD88 mRNA expression
Lactobacillus rhamnosus GG	[28]	1 × 109 CFU/mL immediately before CLP	↓ IL6 and TNF-α mRNA expression and levels ↓ COX-2, TLR-2, MyD88 and NF-κB mRNA expression
	[30]	2 × 109 CFU/mL during 28 consecutive days before CLP	↓ IL-2 and IL-22 levels
	[33]	1 × 109 CFU/mL immediately before CLP	↓ IL-1β, IL-6, TNF-α, and NLRP3 mRNA expression and levels ↓ MCP-1 and NF-κB mRNA expression
Zimomona mobilis	[65]	1 × 109 CFU/mL (twice/day) during 10 consecutive days before CLP	↓ TNF-α levels ↑ IL-10 levels

Interleukin (IL). Monocyte chemoattracctant protein-1/C-C motif chemokine 2 (MCP1/CCL2). Myeloid differentiation primary response 88 (MYD88). Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3). Nuclear Factor-kappaB (NF-κB). Tumor necrosis factor (TNF)-α.

and Figure 1.

It is interesting to note that LGG administration can attenuate multiple organ dysfunction, pro-inflammatory markers, and cell death and regulate imbalances and epithelial disruption in the gut. Despite these findings, four out of five of these studies did not find an efficient response in terms of survival rate. Further research into LGG is required to explore its systemic effects and to establish preclinical protocols, which would allow for the complete recovery from sepsis. Whereas Z. mobilis has functional activity in reducing sepsis-induced lung and spleen injury and mortality, knowledge of the molecular mechanisms and signaling pathways involved in this process are limited. Therefore, this live microorganism could be a promising candidate to prevent an immunosuppressive state because of its ability to abrogate cell death in the spleen, which has a crucial role in sepsis severity. The modulatory effect of Z. mobilis on the long-term complications of sepsis might be addressed in future studies, along with B. longum, which protects the lung tissue during the inflammatory response to systemic infection. Another suggestion addressed in experimental sepsis models involving E. coli or B. subtilis plus E. faecium probiotics, which recover gut barrier functions via the maintenance of tight junction proteins, is to verify if bacterial diversity can be preserved or improved with probiotic supplementation. These preclinical studies suggest that selective probiotics or their combinations might be applied as possible adjuvants for the treatment of sepsis to ameliorate the immune response and regulate inflammation. In this context, further studies on clinical efficacy, safety, costs, and benefits should be conducted.

7. Probiotics in the Pediatric ICU

In addition to animal models, the potentially therapeutic and anti-inflammatory properties of probiotics have been investigated in humans, but their effects on sepsis in clinical settings remain unclear. In 2002, Dani et al. [84] reported that prophylactic LGG administration does not protect newborn and low-birth-weight infants from bacterial sepsis, which has been shown recently with Bacillus clausii treatment [85]. These findings corroborated those of other studies showing that Bifidobacterium breve BBG-001 [86], as well as Lactobacillus and Bifidobacterium, combined with oligosaccharides and lactoferrin [87], were not effective against necrotizing enterocolitis (NEC), sepsis, or mortality in neonates and infants. However, a probiotic combination of Bifidobacterium infantis, Bifidobacterium lactis, and Streptococcus thermophilus was found to protect infants from NEC without positive results for late-onset sepsis [88]. In addition, the adjuvant potential of Saccharomyces spp. in inducing a protective response against sepsis remains unclear because of controversial studies about the efficacy of this treatment [89,90,91].

In contrast, beneficial outcomes were observed with respect to the incidence of late-onset sepsis and inhibition of colonization by Candida strains in the gut with L. rhamnosus and L. reuteri supplementation [92]. Another positive effect of the probiotic combination was a reduction in hospitalization days of low-birth-weight infants [93]. Furthermore, L. plantarum, combined with fructooligosaccharide, promotes a significant reduction in the incidence of culture-positive and culture-negative sepsis and in lower respiratory tract infections in infants [94]. Fortmann et al. [95] recently reported the efficient supplementation of L. acidophilus and B. infantis plus human milk, which protected infants from sepsis. A combination of Lactobacillus and Bifidobacterium strains was found to lower TNF-α, IL-6, IL-12p70, and IL-17 levels and increase IL-10 and TGF-β levels in children with severe sepsis, protecting them from organ failure [96]. Recently, the benefits of probiotic use in preterm infants between 30 and 37 weeks revealed an increase in the feeding capacity and growth and improved gut functions, in addition to reducing the hospital stay [97].

8. Probiotics for Adult Patients in the ICU

The administration of Lactobacillus, Bifidobacterium, and Streptococcus microorganisms along with oligofructose was able to alter the microbiota in the upper gut of adult septic patients, but it had no effect on intestinal permeability or mortality [98]. B. longum, L. bulgaricus, and S. thermophilus increase IL-12p70 and IFN-γ levels in blood and decrease IL-4 and IL-10 levels in patients with severe traumatic-brain injury, reducing the incidence of nosocomial infections and long stays in the ICU [99]. Furthermore, B. breve strain Yakult, L. casei strain Shirota, and galactooligosaccharides administered to septic patients alter the gut microbiota composition and attenuate respiratory complications [100]. However, the mortality rate was not affected in these studies. Finally, some studies suggested that probiotics could be used to reduce sepsis and infectious complications in patients with colorectal liver metastases who had undergone local resection [101] or in post-injury infections in patients with multiple injuries [102] and abdominal surgery [103].

Table 2. Administration of probiotics in children and adults admitted in ICU.

Reference	Number of Subjects Who Received Probiotic Administration	Microorganism	Outcomes
[84]	295	LGG	Sepsis incidence was affected by probiotic administration.
[85]	123	B. clausii	Sepsis incidence was not affected by probiotic administration.
[86]	650	B. breve	Sepsis incidence was not affected by probiotic administration.
[87]	104	Symbiotic preparation with L. rhamnosus, L. plantarum, B. lactis, fructooligosacharide (FOS), galactooligosaccharide (GOS), bovine lactofferin, and vitamins (C, E, B1, B2 and B6).	Sepsis incidence was not reduced by symbiotic mixture administration.
[88]	548	Probiotic combination of B. infantis, B. lactis, and S. thermophilus,	Late-onset sepsis was not reduced by probiotic combination administration.
[89]	91	S. boulardii	The probiotic was able to prevent clinical sepsis and reduce its incidence.
[90]	63	S. boulardii	There was no difference in sepsis incidence.
[94]	2278	L. plantarum plus fructooligosaccharide	The symbiotic preparation was able to reduce sepsis incidence.
[95]	7516	L. acidophilus and B. infantis	The probiotic combination plus human milk was able to prevent sepsis.
[96]	50	L. paracasei, L. plantarum, L. acidophilus, L. delbrueckii, B. longum, B. infantis, B. breve, S. salivarius plus maltose, and silicon dioxide	There was no effect on septic mortality with probiotic administration.
[98]	45	Symbiotic preparation with L. acidophilus, B. lactis, S. thermophilus and L. bulgaricus plus oligofructose.	Symbiotic administration had no effect on septic mortality.
[100]	35	B. breve and L. casei plus galactooligosaccharides.	There were no significant differences in the incidence of bacteremia or septic mortality.
[101]	66	A mixture of L. plantarum, L. acidophilus, and B. longum.	The treatment reduced postoperative septicemia.
[102]	36	A mixture of Pediococcus pentoseceus, L. mesenteroides, L. paracasei ssp. 19, and L. plantarum plus beta glucan, pectin, and resistant starch as bioactive fibers	The treatment was able to reduce the occurrence of postoperative infective complications.

shows the outcomes in sepsis of pediatric and adult patients admitted in the ICU who received probiotic administration.

9. Probiotic Side Effects in Clinical Practice

Even though probiotics have been considered safe to balance the intestinal microbiota, several isolated cases demonstrated that the same strain used to treat gut dysfunctions can translocate into the blood, resulting in septicemia. Saccharomyces cerevisiae and associated variations were mainly reported to be a source of fungemia from newborns to elderly patients [104,105,106,107,108,109,110,111,112,113,114,115,116,117,118]. In addition, bloodstream infections of Lactobacillus spp. [119,120,121,122,123,124,125,126,127,128,129,130,131,132,133], Bifidobacterium spp. [134,135,136,137,138,139], E. coli Nissle 1977 [140], P. pentosaceus [141], and B. clausii [142] have been reported in septic patients following probiotic administration. Interestingly, many of these patients were previously diagnosed with comorbidities [105,112,118,122,125,142], inflammatory disorders [108,113,123,130,140,141], congenital malformations [109,121,127,134,138,139], or acquired immunodeficiency syndrome [124,126,139] before starting probiotic therapy. These relationships can be interpreted as challenging for the use of probiotics in sepsis, since the complexity of the disease itself and its treatment can lead to a similar state of intense inflammation or immunosuppression. Moreover, it is possible that due to underlying diseases, even commensal bacteria or adjuvant supplementation can result in host infection.

10. Conclusions and Future Perspectives

Probiotic supplementary therapy or enteral administration to abolish dysbiosis-related diseases can carefully be evaluated in critically ill patients. First, microbiota composition and establishment are challenged during life. The infant gut microbiota is less stable, more variable, and can be altered by maternal microbiota, lifestyle, health status, complementary food, and duration of lactation. In this period of life, gut microbiota play a key role in immunological network impacting on human health. In the same way, dietary patterns, microbial infections, and clinical interventions can modify adult microbiota composition and increase the risk of diseases [143,144]. In addition, due to the lack of guidelines for probiotic usage in the ICU environment, randomized placebo-controlled trials will help to clarify the efficacy and safety of probiotics to establish a pattern that indicates a cause–effect correlation based on infections and probiotics.

This literature review discussed the potential effects of probiotics as targets for sepsis therapy. Although the majority of the studies reviewed here demonstrated the possibility of using Lactobacillus, Bacillus, and Bifidobacterium strains to manage the complications of sepsis in experimental animal models, the aforementioned clinical trials have focused on testing mainly combinations of probiotics with positive or negative outcomes through partial protection and by modulating cytokines and changing the microbiota composition. Implications regarding the animal model also are addressed. CLP has been used as a preclinical model to clarify the pathophysiology of sepsis and its therapeutic direction. However, different murine strains display distinct susceptibilities to the procedure that can impact on the replication and consistency of results [145,146]. Therefore, the heterogeneous methodology of probiotic administration and their dose ranges, as well as the use of different animal strains and diverse patient profiles, limit our understanding of the possible benefits of clinical probiotic administration.

Prospective multicenter studies with different probiotic strains, alone or in combination, need to be conducted to clarify the effects of probiotics in the hospital environment. Interestingly, many studies have investigated the ability of probiotics to modulate the gut and the hyperinflammatory phase in sepsis. However, the mechanisms of these actions have not been fully elucidated. Additionally, exploring the importance of probiotic treatment to long-term consequences of sepsis in specific tissues, such as lung, kidney, brain, liver, and gut, or to patient readmission to the hospital due to illnesses after recovery from sepsis should reveal strategies that promote physiological homeostasis and protect against post-sepsis syndrome. Finally, further research is necessary to understand whether probiotics could act as immunologic adjuvants that regulate the immune response and sepsis-induced immunosuppression.