**5. Colinfant**

Colinfant is an *E. coli* (A0 34/86) strain that is used as prophylactic in infants for allergy, nosocomial infection, and diarrhea [20, 21]. Additionally, it is effective in later years in preventing infections and developing allergies. Some strains of *Klebsiella oxytoca* are implicated in antibiotic-associated diarrhea, which could be reduced by the administration of Colinfant in infants. Colinfant also prevents infection of pathogenic *E. coli*.

### **6. Genetic modifications of probiotic** *E. coli*

Probiotic *E. coli* strains have been modified to improve colonization, to secrete metabolites, proteins, and enzymes exploiting a variety of genetic manipulations (**Table 2**). EcN was tagged with a green fluorescent protein (Gfp), which facilitated monitoring the colonization and survival in stomach, ileum, colon, and Peyer's patches [36]. EcN was detected in the fecal matter at 45 days after oral inoculation.



*Potential of* Escherichia coli *Probiotics for Improved Health and Disease Management DOI: http://dx.doi.org/10.5772/intechopen.100380*

#### **Table 2.**

*Characteristics of genetically modified probiotic* E. coli *strains.*

EcN contains two cryptic plasmids MUT1 and MUT2, and these plasmids were cured using CRISP-Cas9-assisted double-strand breaks [47]. EcN strain cured of these plasmids had similar growth under Luria broth conditions despite differences in the DNA content. Effects of colonization and survival of the plasmid-cured strain with decreased DNA content as compared to the wild-type strain need to be investigated to determine the impact of metabolic load. Alternatively, both the cryptic plasmids of EcN have been engineered for stable maintenance and expression of recombinant proteins [53].

*Vitreoscilla* hemoglobin (VHb) with a high affinity for oxygen facilitates the survival and functionality of bacteria under microaerobic conditions [54] promoted colonization of genetically modified *E. coli* in the gut. *E. coli* 16 double transformants of *gfp* and *Vitreoscilla* hemoglobin (*vgb*) genes at 108 cfu/g were present in the rat fecal matter after 70 days of oral administration, while Ec16 *gfp* was not found after 48 days [37]. Additionally, catalase activity of VHb scavenges the reactive oxygen species, which decreased the carbon tetrachloride-induced hepatotoxicity in rats.

Pyrroloquinoline quinone (PQQ ) is a water-soluble antioxidant with the highest redox cycles of 20,000, promotes mitochondrial biogenesis and cellular signaling, and provides health benefits [55]. *E. coli* 16 strain tagged with *gfp-vgb* genes and transformed with *pqqABCDE* operon from *Pseudomonas fluorescens* Bf1 prevented colon and liver damage by dimethylhydrazine (DMH) due to the combined beneficial effects of effective colonization and antioxidant properties of Vhb and PQQ, respectively [38]. DMH had systemic oxidative damage, and decreased brain serotonin and norepinephrine levels, but epinephrine levels were increased [39]. In addition to decreasing the oxidative damage, *E. coli* 16 *vgb-pqq* strain had near-normal levels of neurotransmitters in rats. These beneficial effects were not similar with treatments of Ec16, vitamin C, or PQQ alone suggesting other than its additional ability to confer antioxidant properties, probiotic *E. coli* 16 had synergistic effects related to the continuous secretion of PQQ in the gastrointestinal tract. These beneficial effects were also seen in EcN strain that was modified in a similar manner to that of Ec16 strain [40]. EcN *vgb-pqq* recombinant strain effects were monitored in rats for alcohol toxicity in chronic and acute exposure. Chronic alcohol caused extensive oxidative damage and induced hyperlipidemia and the EcN::*vgb-gfp*(*pqq*) probiotic strain prevented the deleterious effects, while EcN, PQQ, and vitamin C alone had no significant effects. These effects were also correlated with increased short-chain fatty acids (SCFA) in the colon. However, oral PQQ had better effects than recombinant EcN strain in acute alcohol damage. These studies further supported the significance of endogenous PQQ biosynthesis by probiotic *E. coli*.

Aging is associated with progressive loss of tissue functions mediated by reactive oxygen species-induced oxidative damage as a result of mitochondrial dysfunction [56–58]. EcN::*vgb-gfp* transformed with *pqq* gene cluster from *Gluconobacter suboxydans* 621 decreased the rotenone-induced mitochondrial oxidative damage in aging rats along with decreased lipogenesis and increased fatty acid oxidation genes correlated with increased colonic SCFA and PQQ in both feces and liver [41]. Additionally, an increase in mitochondrial biogenesis and metabolism indicates delaying of age-related tissue damage.

Heavy metal toxicity is mediated by reactive oxygen species [59]. Chelation of heavy metal ions and antioxidants is used to prevent the toxicity. EcN::*vgb-gfp* strain operon containing *pqq* gene cluster from *Gluconobacter oxydans* decreased the Cd and Hg toxicity upon oral supplementation citric acid due to the antioxidant effects of PQQ and chelation ability of citric acid [42]. Subsequently, EcN::*vgb-gfp* strain containing *pqq* gene cluster from *A. calcoaceticus* and gluconate dehydrogenase (*gad*) operon from *Pseudomonas putida* KT2440 secreted PQQ, gluconic and 2-ketogluconic acids, and this strain prevented toxicity caused by Cd, Hg and Pb without affecting the essential metal ions [43]. Thus, 2-ketogluconic acid produced by EcN recombinant strain is mimicking the chelating abilities of citric acid. Similarly, EcN strain containing As(III) S-adenosylmethionine (SAM) methlyltransferase (*arsM*) and *pqq* gene cluster prevented arsenite toxicity by scavenging arsenite-induced reactive oxygen species by secreted PQQ and converting arsenite into non-toxic trimethylarsenite in rats [44].

EcN recombinant strain containing *pqq* operon secretes 15 mM gluconic acid [43]. Gluconic acid was proposed for cancer therapy as cancer cells utilize citrate for growth and gluconic acid irreversibly inhibits citrate transporter, which is expressed on cancer cells [60]. Hence, EcN producing gluconic acid could prevent the progression of tumors, especially colorectal cancers. *Staphylococcus aureus* α-hemolysin expressing EcN recombinant strain forms pores in the tumor cells resulting in the regression of tumors in mice [51]. Similarly, tumor regression also occurred in mice xenografted with human colorectal cancer cells treated with EcN strain expressing hemolysin E (HlyE) a pore-forming protein [52].

SCFA such as acetate, propionate, and butyrate produced by gut microbiome is necessary for the survival of colonocytes, maintenance of intestinal integrity, mucus production, serotonin release by enterochromaffin cells, and secretion of gut hormone peptide YY in the intestine [61, 62]. Additionally, SCFA also regulates brain and liver functions while diminished SCFA signaling is associated with metabolic diseases [63]. Propionate and butyrate prevent the progression of these metabolic diseases [64]. In order to design EcN to secrete butyric acid, fumarate reductase (*frdA*), lactate dehydrogenase (*ldhA*), alcohol dehydrogenase (*adhE*), and phosphotransacetylase (*pta*) genes involved in the fermentation product

#### *Potential of* Escherichia coli *Probiotics for Improved Health and Disease Management DOI: http://dx.doi.org/10.5772/intechopen.100380*

formation of succinic, acetic, and lactic acids were deleted to generate EcN YF005 strain [49]. The *atoDABE* operon encodes the genes for the formation of acetoacetyl CoA and butyryl CoA to the butyric acid formation, while *hbd* and *crt* from *Clostridium acetobutylicum* and *ter* from *Treponema denticola* genes convert acetoacetyl CoA into butyryl CoA. The native promoter of *atoDABE* operon was replaced with a strong, constitutive PL promoter from phage λ, and synthetic PL-*LacO*-*hpdcrt-ter* operon was integrated at methylglyoxal synthase (*msgA*) gene to generate EcN Y2023 strain. This strain produced 0.49 g/L butyric acid on glucose. It will be interesting to determine its therapeutic potential in animal studies.

EcN deletion mutant of *dapA* gene coding for 4-hydroxytetra-hydropicolinate synthase was generated for incorporating phenylalanine degradation for the treatment of phenylketonuria [48]. Two different SYNB1618 strains were generated by incorporating phenylalanine ammonia-lyase and L-amino acid deaminase (*pma*) genes, which convert phenylalanine into *trans*-cinnamic acid (TCA) and phenylpyruvate, respectively. In humans, TCA is further transformed into hippuric acid in the liver and excreted in the urine. The oral load of 70 mg phenylalanine was reduced by 58% in the serum samples of individuals fed with the modified strain.

EcN was genetically modified for inflammatory bowel disease by probioticassociated therapeutic curli hybrids (PATCH) approach using a fusion protein of amyloid domain for self-assembly (CsgA) linked to trefoil factor-3 with 6 His residues [50]. Oxidatively damaged inflamed regions are conducive for the growth of facultative anaerobes. Consequently, modified EcN strain numbers increased at the damaged regions and secreted curly fibers that facilitated the repair of damaged regions. The EcN-engineered strain could ameliorate the weight loss in DSSinduced colitis in mice.

EcN expressing a fusion protein of cholera toxin B domain and insulin growth factor-1 (CTB-IGF1) was proposed as a long-term therapeutic strategy for diabetes [65]. It was hypothesized that EcN expresses the fusion protein in the intestine that would cross the intestinal epithelium into blood circulation facilitated by CTBspecific interaction with GM1 ganglioside oligosaccharide and IGF will activate the insulin effects.
