The Potential of Probiotics to Eradicate Gut Carriage of Pathogenic or Antimicrobial-Resistant Enterobacterales

Probiotic supplements have been used to decrease the gut carriage of antimicrobial-resistant Enterobacterales through changes in the microbiota and metabolomes, nutrition competition, and the secretion of antimicrobial proteins. Many probiotics have shown Enterobacterales-inhibiting effects ex vivo and in vivo. In livestock, probiotics have been widely used to eradicate colon or environmental antimicrobial-resistant Enterobacterales colonization with promising efficacy for many years by oral supplementation, in ovo use, or as environmental disinfectants. In humans, probiotics have been used as oral supplements for infants to decease potential gut pathogenic Enterobacterales, and probiotic mixtures, especially, have exhibited positive results. In contrast to the beneficial effects in infants, for adults, probiotic supplements might decrease potentially pathogenic Enterobacterales, but they fail to completely eradicate them in the gut. However, there are several ways to improve the effects of probiotics, including the discovery of probiotics with gut-protection ability and antimicrobial effects, the modification of delivery methods, and the discovery of engineered probiotics. The search for multifunctional probiotics and synbiotics could render the eradication of “bad” Enterobacterales in the human gut via probiotic administration achievable in the future.


Introduction
Trillions of bacteria colonize in various anatomical locations in the human body, including the mouth, the upper airways, the skin, the vagina, the genitourinary system, and the intestinal tract. These colonized locations represent a highly integrated ecosystem collectively called "microbiota" [1,2]. Thus, humans are considered to be metaorganisms (also termed superorganisms or holobionts) [1,2]. The overlap of the phylogenetic trees of bacterial microbiota and primates suggests the coevolution, especially the genetic coevolution, between host and microbiota [2][3][4]. The microbial colonization of the human body starts immediately following birth, and the community composition is shaped by

Rationale for Probiotic Supplements to Eradicate Enterobacterales Carriage in the Gut
Probiotics, by definition, are live microorganisms, and should remain viable when they reach the intended site of action, which is typically the cecum and/or the colon [24]. Most probiotics originate from fermenting food, an ancient form of preservation ingrained in human societies around the world [25]. The microbiome of all fermented foods shows increasing amounts of Lactobacillales during the fermentation process, which replaces the initial dominant composition of Enterobacterales in these foods [25]. The incorporation of probiotics into food results in higher counts of lactic acid-producing bacteria and lower counts of Enterobacterales [26]. To date, probiotics have been widely used as food additives.
The eradication of pathogenic Enterobacterales by supplementation with probiotics has been confirmed in several animal models [17][18][19][20]23]. Mice pretreated with B. bifidum ATCC 29521 exhibited a significant increase in the diversity of the gut microbiome, and a decrease in the abundance of the genus Escherichia-Shigella, belonging to the family Enterobacterales [17]. These changes in microbiota after B. bifidum ATCC 29521 pretreatment were associated with a decrease in the severity of inflammatory bowel disease [17]. More-over, L. rhamnosus GG could reduce the mortality rate of septic mice by modulating gut microbiota composition, especially reducing the lipopolysaccharide producers, such as Enterobacterales [18]. Bacillus coagulans SANK 70258 suppressed Enterobacterales and enhanced butyrogenesis in microbiota models [19]. L. plantarum, isolated and identified from yak yogurt, increased the content of beneficial bacteria, including Bacteroides, Bifidobacterium, and Lactobacillus, and reduced the content of harmful bacteria, including Firmicutes, Actinobacteria, Proteobacteria, and Enterobacterales, and, thus, could protect against alcoholic liver injury [20]. The oral administration of L. rhamnosus GG can improve the survival rate of mice with sepsis by reducing lipopolysaccharide-producing Enterobacterales, decreasing epithelial apoptosis, and increasing the proliferation of colonic epithelium and the expression of tight junction proteins [23]. A mixture of probiotics showed more efficient eradication of pathogenic Enterobacterales in vivo. In mice, the mixture of L. fermentum GOS47 and L. fermentum GOS1 significantly decreased the viable count of Enterobacterales with potential anti-inflammatory activity and short-chain fatty acid production [27]. Thus, the favorable effect of probiotic supplements on at least the partial elimination of pathogenic Enterobacterales, ex vivo and in vivo, has promoted their application in clinical diseases.
Supplementation with probiotics has been investigated for the alleviation of the disease severity of systemic or gastrointestinal inflammatory diseases, such as sepsis, inflammatory bowel disease, and chemotherapy-or radiation-induced gastrointestinal mucositis [17][18][19]28,29]. For example, patients receiving cytotoxic and radiation therapy showed striking alterations in intestinal microbiota with, most frequently, a decrease in Bifidobacterium, Clostridium cluster XIVa and F. prausnitzii, and an increase in Enterobacterales and Bacteroides [28]. These pathogenic alterations resulted in the development of mucositis and bacteremia [28,29]. The prevention of cytotoxic chemotherapy-induced mucositis by probiotics has been investigated in randomized clinical trials with some promising results. Moreover, in a meta-analysis of randomized controlled trials with patients undergoing a colorectal resection, the perioperative administration of probiotics or synbiotics was associated with increased numbers of Lactobacillus and decreased counts of Enterobacterales [30]. These changes in gut microbiota were associated with less diarrhea, less symptomatic intestinal obstruction, and a lower incidence of total postoperative infections [30]. Accordingly, the use of probiotics in modulating gut microbiota and decreasing pathogenic Enterobacterales has become popular for application in many bowel or extra-bowel diseases, and more extensive probiotic usage can be expected in the future.

Probiotic Supplements to Decrease Gut Carriage of Enterobacterales in Livestock or Domesticated Animals
The use of probiotics in preventing gut Enterobacterales colonization has been applied in livestock breeding [31][32][33][34][35][36]. Lactobacillus supplementation, in directly fed microbes or used as phytobiotic feed additives, reduced the prevalence of ESBL-producing Enterobacterales in broilers [31]. In young broilers, the neonatal colonization of Enterobacterales strains led to immune dysregulation and chronic inflammation, but early life exposure to a mixture of probiotics containing lactic-acid-producing bacteria could modulate the immune functions through the activation and trafficking of immune cells [32]. In weaned piglets, B. subtilis DSM25841 treatment reduced enterotoxigenic E. coli (ETEC) F4 infection and decreased the risk of diarrhea [34]. L. reuteri KUB-AC5 possessed antimicrobial activity in reducing Salmonella contamination in live poultry [35]. The above data further support the use of probiotics as feed additives in livestock breeding.
Other than oral intake, the in ovo administration of probiotics for eradicating gut Enterobacterales colonization has been used in chickens [37,38]. Via the in ovo route during hatching, a Bacillus-based probiotic (BPP) can reduce the severity of the virulent E. coli horizontal transmission among broiler chickens, which might be achieved by alterations in the microbiota composition, including a decrease in Enterobacterales and an increase in Lachnospiraceae [37]. In another chicken study, the in ovo administration of lacticacid-producing bacteria resulted in an increased abundance in the Lactobacillaceae family and Lactobacillus genus, and a decrease in Enterobacterales and Enterococcaceae [38]. For bird species, the early in ovo administration of probiotics seems to be more efficient in eradicating gut Enterobacterales colonization before hatching.
A mixture of probiotics may work better to eradicate gut Enterobacterales in livestock breeding [33,[39][40][41]. The administration of multistrain probiotics containing L. acidophilus LAP5, L. fermentum P2, Pediococcus acidilactici LS, and L. casei L21 could modulate intestinal microbiota (increase Lactobacillaceae abundance and reduce Enterobacterales abundance), increase the gene expression of tight junction proteins (ZO-1 and Mucin 2) and the immunomodulatory activity (downregulation of mRNA levels of interferon-γ [IFN-γ] and lipopolysaccharide-induced tumor necrosis factor-α [TNF-α], and upregulation of IL-10) in broiler chickens [33]. Commercially available synbiotics, either BioPlus 2B ® or Cylactin ® LBC, had a more significant impact on the concentration of lactic acid, short-chain fatty acids (SCFAs), and branched-chain fatty acids (BCFAs), than a single probiotic in sows [39]. Mixed probiotics composed of three thermophilic lactic-acid-producing bacteria (LAB) strains, L. helveticus BGRA43 (strong proteolytic activity, antimicrobial activity, and adhesion to gut cell activity), L. fermentum BGHI14 (immunomodulatory effect), and Streptococcus thermophiles BGVLJ1-44 (strong proteolytic activity and immunomodulatory effect), influenced the colonization of piglet guts with beneficial bacteria, and reduced the number of Enterobacterales in some treated sows [41]. Thus, the commercially available mixed regimens of probiotics may be more efficient in eliminating Enterobacterales carriage in the guts of livestock.
Furthermore, probiotics in combination with prebiotics (foods that promote the growth of beneficial microbes), or phytobiotics (plant-derived products), have been utilized in livestock breeding for the eradication of gut colonization by Enterobacterales [40,42]. Lactobacillus strains (L. agilis and L. salivarius), combined with phytobiotics, have been used to reduce the survival of potentially problematic bacteria, such as ESBL-producing E. coli in broilers [42]. The synbiotics (L. rhamnosus HN001 and P. acidilactici) combined with the phytobiotics (Agave tequilana fructans) induced morphological modifications in the duodenal mucosa of broilers that, in turn, promoted resistance to infections caused by S. typhimurium and C. perfringens [40].
In addition, a probiotic-based cleaning strategy to decontaminate Enterobacterales in livestock environments has been reported [43]. The cleaning product, containing B. subtilis, B. pumilus, and B. megaterium spores, was used to clean fresh and reused broiler litters [43]. These Bacillus spores were able to successfully colonize reused poultry litters to decrease the mean counts of total aerobic bacteria, Enterobacterales, and coagulasepositive Staphylococcus [43]. A decrease in Enterobacterales, mainly the genus Escherichia, was also observed in the ceca of broilers reared on reused litters treated with the cleaning product [43]. The efficacy and safety issues of this probiotic-based cleaning product are still ongoing for livestock environments, but have not been tested for human environments.
Among dogs fed Queso Blanco cheese with B. longum KACC 91563 for eight weeks, a reduction in harmful bacteria, such as the Enterobacterales and Clostridium species, was noted [22]. The successful decrease in Enterobacterales after probiotic supplementation in pet animals arouses hope for the eradication of gut Enterobacterales carriage via the use of probiotics in humans.

The Selection of Probiotics to Decrease Gut Colonization of Enterobacterales in Humans
The common, safe, and well-studied probiotics used to eradicate the gut carriage of Enterobacterales in humans include the Lactobacillus [44][45][46][47] and Bifidobacterium [17,47,48] species. In extremely low-birth-weight infants, L. reuteri supplementation for one week resulted in a lower abundance of Enterobacterales and Staphylococcaceae [44]. Among infants fed B. infantis EVC001, a high abundance of Bifidobacteriaceae developed rapidly with a reduced abundance of antibiotic-resistant genes among Enterobacterales and/or Staphylococcaceae [48].
As noted in livestock, probiotic mixtures might provide better protection against gut Enterobacterales colonization than a single probiotic regimen in humans [45,[49][50][51]. A probiotic mixture (Bactiol duo ® ) containing Saccharomyces boulardii, L. acidophilus NCFM, L. paracasei Lpc-37, B. lactis Bl-04, and B. lactis Bi-07, provides better eradication of AmpCproducing Enterobacterales carriage than S. boulardii CNCM I-745 ® [45]. Oral daily supplementation with a combination of a prebiotic (Emportal ® : lactitol) and probiotics (Infloran ® : B. bifidum and L. acidophilus) for three weeks decreased the intestinal load of OXA-48producing Enterobacterales among eight patients with long-term intestinal carriage [49]. Moreover, the ingestion of combined probiotics containing L. plantarum LK006, B. longum LK014, and B. bifidum LK012 could significantly reduce the abundance of Enterobacterales and increase the abundance of Lactobacillaceae in preterm infants [50]. These changes in microbiota were correlated with a decreased serum inflammatory cytokine level of IL-6 and improved the survival rate of these infants. A mixture of B. breve M-16V, B. longum subsp. infantis (B. infantis) M-63, and B. longum subsp. longum BB536, achieved significantly higher levels of Bifidobacterium-predominant microbiota and lower detection rates for Clostridium and Enterobacterales than a single B. breve strain [51]. For human safety, the most common probiotics for combination are the Lactobacillus and Bifidobacterium species.

Probiotic Supplementation to Decrease Potential Gut Pathogenic Enterobacterales from Infants to Children
Probiotics have been used as supplements for infants to decease potential gut pathogenic Enterobacterales [44,48,50,[52][53][54][55][56][57] (Table 1). Among hospitalized infants, early administration of L. reuteri DSM 17938 was associated with less colonization by diarrheagenic E. coli [55]. In a randomized placebo-controlled study that administered B. infantis to 24 infants with gastroschisis, the microbial communities were not significantly influenced [52]. In a doubleblind, placebo-controlled randomized clinical study conducted on 69 preterm infants, B. lactis BB-12 supplementation resulted in lower viable counts of Enterobacterales [57]. Moreover, in a randomized trial of 300 healthy newborns, the receipt of B. longum BB536 was associated with a higher Bifidobacterium/Enterobacterales ratio (B/E), an increased number of IFN-γ-secreting cells, and a higher ratio of IFN-γ/IL-4-secreting cells, which is indicative of the increased Th1 response [54]. Among 21 neonates that underwent surgery for congenital heart disease >7 days after birth, the enteral B. breve strain Yakult (BBG-01) supply led to significantly fewer Enterobacterales in the gut [56]. Since infants, especially preterm infants, are susceptible to intestinal infection, many probiotic studies have been conducted on these susceptible hosts that have provided promising results against pathogenic Enterobacterales colonization in the gut.
However, not all studies have shown the presence of the beneficial effects of the addition of probiotics for infants. In a double-blind randomized control trial, 21 bottle-fed preterm infants receiving L. rhamnosus GG did not show a decrease in the numbers of Enterococcus and Enterobacterales in the gut, increased weight gain, or a decreased hospital stay compared to 26 control infants [58]. In an early review of randomized controlled trials including preterm infants, the B. animalis subsp. lactis supplement could increase fecal Bifidobacterium counts and reduce Enterobacterales and Clostridium counts, but it did not influence the risk of necrotizing enterocolitis or sepsis [59]. The diverse inhibitory potential of Enterobacterales, and the microbiota-modulating effect of probiotics, are likely due to the intrinsic diversity of the gut microbiota of infants and children inhabiting different areas [60].

Probiotic Supplementation to Decrease Gut Pathogenic or Antimicrobial-Resistant Enterobacterales Colonization in Adults
Among adults, probiotic supplements have been shown to decrease, but have failed to totally eradicate, potential antimicrobial-resistant or pathogenic Enterobacterales in the gut [45,47,[61][62][63][64][65][66] (Table 2). To eradicate potential antimicrobial-resistant Enterobacterales, a clinical trial of a probiotic mixture (Bactiol duo ® : S. boulardii, L. acidophilus NCFM, L. paracasei Lpc-37, B. lactis Bl-04, and B. lactis Bi-07) showed that colonization with AmpCproducing Enterobacterales transiently increased after amoxicillin-clavulanate therapy and declined after probiotic intervention [45]. To eradicate potential pathogenic Enterobacterales in human-immunodeficiency-virus-infected individuals, L. rhamnosus GG supplementation was used and resulted in a decrease in intestinal inflammation, along with a reduction in Enterobacterales in the gut [62]. The consecutive intake of fermented soymilk (containing isoflavone) and L. casei Shirota among 60 healthy premenopausal Japanese women was able to decrease the fecal levels of Enterobacterales and to increase isoflavone bioavailability [63].
In contrast to the promising results of the probiotic trials on the eradication of potential antimicrobial-resistant Enterobacterales mentioned above, a randomized single-blind, placebo-controlled trial in southern Sweden used a probiotic mixture of eight living bacterial strains, Vivomixx ® , but the successful eradication of fecal ESBL-producing Enterobacterales carriage was rarely observed [47]. Among 31 Danish adults who traveled to India for 10-28 days, the ingestion of L. rhamnosus GG had no effect on the risk of ESBL-producing Enterobacterales colonization [61]. Of note, in 75 patients who underwent elective colon surgery, the oral intake of L. plantarum 299v for one week resulted in increased Enterobac-terales and Gram-negative anaerobes in the colon, but no change in the incidence of bacterial translocation or postoperative complications [66]. The diverse effect of probiotic supplements on gut Enterobacterales carriage is likely due to the different baseline gut microbiota and the decolonization efficacy of a variety of probiotic components. To date, probiotic supplementation is not routinely recommended to replace routine antibiotic decontamination in the preoperative preparation of the digestive tract [67]. However, probiotics or synbiotics might be used in combination with a conventional bowel preparation to reduce the fecal carriage of Enterobacterales [68]. However, the majority of larger-scale clinical trials show no evident clinical benefits, such as lower inflammatory responses, fewer infectious complications, or higher survival rates, among adults who consume probiotic supplements. Colonization with AmpC-producing Enterobacterales declined after the probiotic intervention

Possible Mechanisms by Which Probiotic Supplementation Decreases Gut Enterobacterales Carriage
The decrease in gut Enterobacterales carriage after probiotic supplementation might be related to the increase in beneficial bacteria, such as Bacteroides, Bifidobacterium, and Lactobacillus [20,48,50], and SCFA-producing bacteria (such as Lachnospiraceae and Ruminococcaceae) [69], as well as to the changes in the metabolome in the gut (Figure 1) [39,70]. The continuous intake of a combination of probiotic cheese enriched with L. reuteri CCM 8617RIF and crushed flaxseed resulted in an alleviation of the infection course induced by pathogenic E. coli O149:F4NAL, favored n-3 polyunsaturated fatty acid metabolism, and inhibited n-6 PUFA metabolism in the gut [70]. Moreover, a probiotic mixture resulted in a greater increase in lactic acid, SCFAs, and branched-chain fatty acids (BCFAs), than a single probiotic in sows [39]. Conclusively, the gut metabolome changes after probiotic supplementation builds up an environment that is not friendly to Enterobacterales. In addition, a microbe is regarded as a beneficial probiotic if it can secrete antimicrobial proteins targeting pathogenic Enterobacterales. For example, a probiotic strain, E. coli Nissle 1917, secretes small proteins called microcins that possess antimicrobial activity against pathogenic Enterobacterales during intestinal inflammation [71]. Therefore, probiotics capable of producing antimicrobial proteins might provide better Enterobacterales eradication efficacy. The Enterobacterales eradication capacity of probiotics can also result from the nutrition competition between probiotics and Enterobacterales. Commensal microbiota contributes to colonization resistance by competing with Salmonella enteritidis for oxygen, a resource critical for pathogen expansion [72]. An analysis of the complex nutrition competition in the microbiota of the gut provides an alternative method for selecting appropriate probiotics against Enterobacterales.

Improve the Effect of Probiotics in Eradicating Enterobacterales
Changes in the delivery method of probiotics might provide alternative ways of eradicate Enterobacterales in the gut [73]. Among patients with mild left-sided ulcerative colitis, the oral intake of L. casei DG failed to affect colonic flora, but the rectal administration of the same probiotics increased Lactobacillus spp. and reduced Enterobacterales, significantly decreased Toll-like receptor (TLR)-4 and IL-1β mRNA levels, and increased mucosal IL-10 [73]. For probiotics vulnerable to gastric acid or intestinal enzymes, rectal administration might provide better efficacy for eradicating Enterobacterales in the gut.
Other than naturally found probiotics, engineered probiotics specifically targeting Enterobacterales have been investigated to improve gut colonization eradication. The introduction of genes with beneficial effects into probiotics provides additional effects, such as acid resistance, immune modulation, and gut barrier protection effects. A genetically engineered plasmid was delivered to E. coli that gained the capacity to produce tetrathionate which can inhibit the growth of Salmonella [74].
To facilitate the buildup of healthy gut microbiota, the ex vivo selection of appropriate probiotics is mandatory. Bacteriocin-producing bacteria capable of inhibiting bovine and wastewater E. coli isolates have been tested for their activity against Shiga toxin-producing E. coli, antimicrobial-resistant E. coli, and related enteric pathogens [75]. The selected bacteriocin-producing bacteria show potential as next-generation control strategies in livestock and humans. Another selected probiotic is B. infantis, a unique gut bacterium with a prodigious capacity to digest human milk oligosaccharides, that was specifically selected for the focused manipulation of infant intestinal microbiota [76].

Clinical Trials of Probiotics or Synbiotics to Improve Gut Health
There are three ongoing randomized clinical trials on dietary supplementation with probiotics aimed at gut Enterobacterales eradication registered at ClinicalTrials.gov, posted from July 2008 to July 2021 (Table 3) [77]. Commercial probiotic mixtures, synbiotics, are being applied in these three trials, and the commonly used probiotic strains include the Lactobacillus, Bifidobacterium, and Streptococcus species. Of note, one interesting trial is comparing the effects of gut Enterobacterales eradication between synbiotics and fecal microbiota transplantation.

Clinical Safety Issue of Probiotics
Although the efficacy of probiotics in decreasing the gut carriage of Enterobacterales has been recognized, there are still concerns regarding their clinical safety, including potential infections or the inflammatory/fatal effects derived from toxins produced either by the probiotic strains or bacterial contaminants [78]. Lactobacillus infections after taking probiotic products containing the Lactobacillus species have been reported in immunocompromised patients [79][80][81]. Lactobacillus endocarditis has been reported in an otherwise healthy patient taking a probiotic formulation containing Lactobacillus [82]. Moreover, there are substantial concerns about the transfer of resistance genes among probiotics, pathogens, and gut microbiota through horizontal gene transfer and the adverse potential of probiotics as the source of antimicrobial resistance genes [83,84]. Thus, the clinical application of probiotics for decreasing Enterobacterales gut carriage among patients with an immunocom-promised status should consider the possibility of opportunistic infections caused by these probiotic strains.

Conclusions
The rationale for using probiotic supplements to eradicate gut Enterobacterales carriage includes changes in the microbiota and metabolomes, nutrition competition, and the secretion of antimicrobial proteins to establish a gut environment that is not friendly to Enterobacterales. Many probiotics indeed do show Enterobacterales-inhibiting effects ex vivo and in vivo. In livestock, probiotics have been widely used to eradicate colonic or environmental Enterobacterales colonization for years, either administered by oral supplementation, in ovo use, or used as environmental disinfectants. For humans, probiotics have been used as dietary supplements for infants to decease potentially pathogenic Enterobacterales in the gut, and probiotics mixtures have shown promising results. This encouraging effect of probiotics on decreasing the gut carriage of Enterobacterales is likely related to the simple gut microbiota in infants, and less interference from underlying chronic diseases and prior antimicrobial exposure.
In contrast to the beneficial effects in infants, for adults, probiotic supplements might decrease potentially pathogenic Enterobacterales in the gut, but they fail to eradicate them. More efforts to confront dysbiosis resulting from comorbidities or antimicrobial therapy, and to select multifunctional probiotics or synbiotics to improve gut health in elderly patients with complex health problems, are currently required.  Institutional Review Board Statement: The approval by Institutional Review Board is waived due to the article nature of review article.