Singh 2021

Critical Reviews in Microbiology
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/imby20
Next-generation probiotics: a promising approach

towards designing personalized medicine
Tejinder Pal Singh & Basavaprabhu Haranahalli Natraj
To cite this article: Tejinder Pal Singh & Basavaprabhu Haranahalli Natraj (2021): Next-generation
probiotics: a promising approach towards designing personalized medicine, Critical Reviews in
Microbiology, DOI: 10.1080/1040841X.2021.1902940
To link to this article: https://doi.org/10.1080/1040841X.2021.1902940
Published online: 06 Apr 2021.
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CRITICAL REVIEWS IN MICROBIOLOGY
https://doi.org/10.1080/1040841X.2021.1902940
REVIEW ARTICLE
Next-generation probiotics: a promising approach towards designing

personalized medicine
Tejinder Pal Singha and Basavaprabhu Haranahalli Natrajb
a
Dairy Microbiology Department, College of Dairy Science and Technology, Lala Lajpat Rai University of Veterinary and Animal
Science, Hisar, India; bDairy Microbiology Division, ICAR-National Dairy Research Institute, Deemed University, Karnal, India
ABSTRACT ARTICLE HISTORY

Second brain, forgotten organ, individual’s identity card, and host’s fingerprint are the few col- Received 17 April 2020
lective terms that are often used to describe the gut microbiome because of its variability, Revised 1 November 2020
accountability, and its role in deciding the host’s health. Also, the understanding of this host Accepted 9 March 2021
health–gut microbiota relationship can create an opportunity to control an individual’s health by Published online 29 March
2021
manipulating the gut microbiota composition. Several approaches like administration of pro-
biotic, prebiotics, synbiotics, faecal microbiota transplantation have been tried to mitigate the KEYWORDS
dysbiosis originated ill effects. But the effects of these approaches are highly generic and non- Next-generation probiotics;
specific. This creates the necessity to design personalized medicine that focuses on treatment of personalized medicine;
specific disease considering the individual specific gut microbiome. The health promoting com- designer probiotics;
mensals could be the new promising prophylactic and therapeutic agents for designing person- gut microbiome
alized medicine. These commensals are designated as next-generation probiotics (NGPs) and
their unusual characteristics, unknown identity and special growth requirements have presented
difficulties for researcher, industrial exploitation, and regulatory agencies. In this perspective, this
review discusses the concept of NGPs, NGP candidates as tool for designing personalized medi-
cine, designer probiotics as NGPs, required regulatory framework, and propose a road map to
develop the NGP based product.
Introduction probiotics such as designer and next-generation probi-

otics (NGPs). The milestones in the history of probiotics
Although the recent advances in molecular biology and
and the change in probiotics concept have been
bioinformatics has empowered the scientists to charac-
depicted in Figure 1.
terize the composition and function of the human gut
Etymologically, the term probiotic is a composite of
microbiome, but the microbial association with human
Latin preposition “pro” meaning “for” and Greek adjec-
gut, their health promoting potential and successful
tive “biotikos” meaning “fit for life”. It was first used by
application has a past of centuries. In 1889, Tissier iso-
Kollath (1953) for the description of various organic and
lated bifidobacteria for the first time from breastfed inorganic supplements that can restore the health of
infant gut but the concept of altering gut microbiota malnourished patients, which later was used by various
for promoting health was introduced by Elie researchers for distinct descriptions. Finally, the restric-
Metchnikoff (1907), who suggested that long life span tion of the term probiotic to viable organisms was pro-
of Bulgarians owing to regular consumption of lactic posed by Fuller (1989). The expert panel convened by
acid bacteria-rich fermented yogurt. In 1930, Minoru the International Scientific Association of Probiotics and
Shirota started the commercialization of probiotics Prebiotics (ISAPP) grammatically updated the probiotics
drink (Yakult) containing Lactobacillus casei strain definition given by FAO/WHO which now is read as “live
Shirota (O’Toole et al. 2017). Thereafter, the inclination microorganisms that, when administered in adequate
of stakeholders (researchers, industries, regulators, and amounts, confer a health benefit on the host” (Hill
consumers) towards objectives, concepts, and interests 2014). The commercial probiotics are mainly
of probiotics have perpetually switched from fermented Lactobacillus spp., Bifidobacterium spp., and many
foods to conventional probiotics to advanced modern others such as Saccharomyces, Bacillus spp., Escherichia
CONTACT Tejinder Pal Singh 88tejindersingh@gmail.com Dairy Microbiology Department, College of Dairy Science and Technology, Lala Lajpat Rai
University of Veterinary and Animal Sciences, Hisar 125004, India; Basavaprabhu Haranahalli Natraj basavaprabhu73@gmail.com Dairy
Microbiology Division, ICAR-National Dairy Research Institute, Karnal 132001, India
ß 2021 Informa UK Limited, trading as Taylor & Francis Group
2 T. P. SINGH AND B. H. NATRAJ
Figure 1. Changing trends and milestones in the history of probiotics.
coli, Enterococci, and Weissella spp. which were selected promoting and disease-fighting roles of probiotics,
either randomly from the gut or traditional fermented there have been a handful of limitations that are ham-
foods that have a long history of safe use. Although pering the applications of conventional probiotics. Of
several investigators have reported the health- note, probiotics administration mainly focussed only on
CRITICAL REVIEWS IN MICROBIOLOGY 3
general effects which include enhancement of intestinal the life-span. At infancy, the gut microbiota compos-
epithelial barrier function, elevation in IgA levels in ition is relatively lucid and has a characteristic abun-
intestinal fluids, maintenance of gut microbiota homeo- dance of Akkermansia muciniphila, Bacteroides,
stasis, reduction of pathogenic organisms in the gut by Veillonella, Clostridium coccoides spp., and Clostridium
the production of antimicrobial components, and pro- botulinum spp. The composition varies with the mode
duction of essential molecules (Okumura and Takeda of delivery, birth gestational date, type of feed, chemo-
2018). Henceforth, probiotics administration does not therapy, or antibiotic use. The gut microbiota becomes
aim at the amelioration of targeted disease. Also, the highly diverse yet relatively stable in adulthood and
efficacies of the conventional probiotics are statistically mainly dominated by Firmicutes (Lachnospiraceae and
marginal because the effects are very much strain-spe- Ruminococcaceae), Bacteroidetes (Bacteroidaceae,
cific and the effects may also vary among the host Prevotellaceae, and Rikenellaceae), and Actinobacteria
(Barzegari and Saei 2012). (Bifidobacteriaceae and Coriobacteriaceae) but host-
Personalized medicine is an advanced approach that specific uniqueness is attributed to genetics, environ-
accounts for the variability including genetic, environment, diet, lifestyle, and gut physiology. In the older
mental, and lifestyle among individuals and character- stage, a reduction in bifidobacteria count and increase
izes the unique complex disease-specific metabolic in Clostridium spp. and Proteobacteria have been
patterns of each patient (Savoia et al. 2017). This futur- observed owing to changing dietary habits, digestion,
istic approach is based on routine analyses of an indi- nutrient absorption, and weak immune system.
vidual’s microbiome and the predictive response of an Evidences from the host–microbe interaction studies
individual to the different nutrients and therapeutic have shown the regulatory function of the gut micro-
agents, thereby, creating an opportunity to develop biota as a crucial moderator that decides the health sta-
novel disease-specific therapeutic strategies. tus of the host (Feng et al. 2018). Studies on germ-free
Considering these points, NGPs and the development (GF) animals proved that gut microbiota has control
of designer probiotics may represent a reasonable over metabolic functioning and appetite signalling in
benign strategy in an era of personalized medicine that the host, intestinal epithelial barrier integrity that func-
can be used to reshape the gut microbiome and, tions to protect the host against pathogens, and devel-
thereby, helps ameliorate the target diseases. opment of host’s immune system (Wang et al. 2017).
Studies have also shown that gut microbiota dysbiosis
may lead to various gastrointestinal (GI) or non-GI infec-
Human microbiota: source of potential next-
tions. Currently, the administration of dietary supple-
generation probiotics
ments like probiotics, prebiotics, or synbiotics is one
Humans are recognized as “Superorganisms” but they strategy that helps to restore the gut homeostasis but
are not alone as they are closely associated with the effects are highly generic and not disease targeted.
microbes. Human microbiome project excavated the Therefore, the advanced approach like personalized
presence of 100 trillion microorganisms (bacteria, fungi, medicine is focussing on individual gut microbiota sig-
archaea, viruses, and protozoa) within the human nature that influences the immune and metabolism
gastrointestinal tract (GIT) which is referred to as gut functions. Personalized medicine aims to reinforce the
microbiome. The GIT is densely populated with bacteria positive influences of the gut microflora on different GI
and culturing all of them is perhaps a difficult task due and non-GI infections through newly recognized routes
to lack of prior knowledge on nutritional requirements of communication such as gut–brain axis, gut–lung
with appropriate culture condition techniques. This axis, gut–bone axis, gut–vascular axis, and other axes
paucity has been overcome with the advent of nucleic (Feng et al. 2018).
acid sequencing techniques and bioinformatics tools Numerous gut microbiota studies and the success
which have allowed the researchers to identify and/or rates of faecal microbiota transplantation (FMT) in the
quantify the different members of the gut microbiota. treatment of recurrent Clostridioides difficile (rCDI) infec-
Phylogenetically, approximately 90% bacterial population and restoration of microbiota dysbiosis have
tion of the gut bacteria belongs to two phyla underlined the importance of commensal species in
Firmicutes and Bacteroidetes while others belong to maintaining individual’s health (Petrof and Khoruts
phyla Actinobacteria, Proteobacteria, Fusobacteria, and 2014). Nevertheless, the success of FMT failed to over-
Verrucomicrobia (Arumugam et al. 2011). Rinninella shadow the associated difficulties like microbiome opti-
et al. (2019) reviewed that every individual has unique mization for treatment, safety assurance of the
gut microbiota composition which varies throughout recipient, and monitoring of patient’s faecal
Table 1. Signature characteristics of conventional probiotics and next-generation probiotics.

Conventional probiotics NGP
Isolated from gut, breast milk, and fermented foods. Mostly derived from commensals.
Belong to limited genera mainly Lactobacillus spp. and Identified from comparison results between healthy and disease
Bifidobacterium spp. animals/people.
Long history of use. NGS/microbiota research platforms (multi-omics study) used.
Generally regarded as safe at the strain level by the US FDA or as Belong to diverse genera.
qualified presumption of safety (QPS) at the species level by EFSA.
Reckoned being safe; as food or food supplements. Strict safety and regulations required.
Do not include genetically modified microbes and are target to general Include genetically modified bacteria and are targeted to a
sub-health population people. specific disease.
Other genera include Streptococcus spp., Bacillus spp., E. coli, and S. Multi-faced information (mechanisms) required
cerevisiae (yeast), etc. Individual strains may function vary
Included in categories of food additives to LBP (drugs)
modification success. Indeed, such limitations of avail- unmodified organisms. Whereas, others believe in the
able approaches have created opportunities for the extensive use of term NGP as it includes the microor-
emergence of more promising approaches. A shift from ganisms that will confirm LBP including genetically
conventional probiotics to the development of NGPs modified bacteria as well as potential beneficial com-
for designing personalized medicine is one such mensal bacteria. It is also believed that with the rapid
upcoming promising and safer strategy that can be advancements in understanding soon the NGPs will
employed to cure target/specific disease. merge either with current probiotics or with LBPs
(O’Toole et al. 2017).
NGPs vs. conventional probiotics
NGPs as personalized medicine
Considering the importance of gut microbiota, the
identification of key players from the commensals that In past decades, the gut microbiota has emerged as a
can address specific health issues could be the new biomarker to describe the health state, prognosis, and
cusp in the probiotics arena. These commensals are to predict the efficacy of the therapy (Kho and Lal
generally referred to as NGPs. NGP is considered a rea- 2018). The association of gut microbiota dysbiosis with
sonable attempt to mark the succession from trad- GI and non-GI diseases is evident from the microbiome
itional microorganisms with safe history to new wide association studies. As there is variability in the
microorganisms with no available evidences. The other presentation, location, therapeutic strategies, and diag-
distinctions between NGPs and conventional probiotics nostic approaches for each disease; the responses to
are depicted in Table 1. NGPs are also sometimes particular treatment therapy may not be the same in all
termed as live biotherapeutic products (LBPs) but it is patients. Therefore, the concept of personalized medi-
also worth considering these terms as different. As per cine that targets diseases considering the patient’s spe-
the US Food and Drug Administration (FDA), LBP is a cificity needs to be adopted. NGPs appeared as a
biological product that (a) contains live organisms, such potential tool for personalized medicine as it may be
as bacteria, yeast (except filterable viruses, oncolytic used to ameliorate the target disease by modulating
bacteria, or products intended as gene therapy agents), gut microbiota. There are two strategies commonly
(b) is applicable to the prevention, treatment, or cure of employed for the development of NGPs. First includes
a disease or condition of human beings, and (c) is not a identification of the strain associated with a particular
vaccine, and (d) is not administered by injection. health phenotype and validating its efficacy to recapitu-
Therefore, NGPs also fits well in the category of LBPs late the health phenotype using appropriate experi-
which include the LBP microorganism as well as the mental models. The second strategy includes the
other components. NGPs can be studied by laboratories identification of an effective molecule that can abro-
already involved in conventional probiotic and micro- gate the disease phenotype and its introduction in a
biome research and can utilize probiotic experience for well-characterized probiotic strain, which may serve as
development trajectory, whereas, LBPs are dealt by a delivery vehicle.
start-up biotechnology companies or pharmaceutical Different bacterial strains have been identified and
companies with marketing interest (O’Toole et al. 2017). studied as NGPs for the amelioration of diseases like
There are contradictory opinions on LBPs among obesity, cancer, pathogenesis, inflammatory bowel dis-
researchers since some believe that genetically modi- ease (IBD), etc. as given in Table 2. Bacteroidetes and
fied probiotics must be dealt under the label domain as Firmicutes are the most dominating phyla in the gut
it satisfies the consumer confidence that probiotics are microbiome, and thereby, the species form these
Table 2. Key features and effects of different NGP candidates studied.
Family Genus and species Genome size; GC content Key features Effects studied References
Phylum: Actinobacteria
Bifidobacteriaceae Bifidobacterium longum 2.48 Mbp; 59.8% Gram-positive, non-motile, often branched Anti-tumour effect in mice model Sivan et al. (2015)
Bifidobacterium breve 2.31 Mbp; 58.9% anaerobic bacteria, possesses a unique Prognostic effect in patients with HBV- Rong et al. (2017)
fructose-6-phosphate related hepatocellular carcinoma
Bifidobacterium lactis 1.93 Mbp; 60.49% phosphoketolase pathway Amelioration of CRC in the rat- Le Leu et al. (2010)
azoxymethane model
Improved immune function in Roller et al. (2007)
polypectomized and colon cancer patients
Eggerthellaceae Gordonibacter 3.29 Mbp; 66% Gram-positive, obligate anaerobe, short-rod Anti-inflammatory; anti-carcinogenic; Selma et al. (2017)
urolithinfaciens bacilli, non-spore former, catalase-positive, cardioprotective;
Gordonibacter 3.6 Mbp; 64% oxidase-negative, urolithin-producing neuroprotective properties
pamelaeae
Coriobacteriaceae Collinsella aerofaciens 2.30 Mbp; 60.1% Gram-positive, rod-shaped, non-motile, Enhanced effect of anti-PD-1 antibody Matson et al. (2018)
obligate anaerobe, ferment a range of therapy in metastatic melanoma patients
plant and animal origin carbohydrates
and for producing H2, ethanol, short-
chain fatty acids, and lactate, major
utilizer of lactose in human colon
Phylum: Firmicutes
Ruminococcaceae Faecalibacterium 2.86 Mbp; 56.9% Gram-positive, rod-shaped, obligate Amelioration of inflammatory bowel disease Sedwick (2014)
prausnitzii anaerobe, butyrate, and other in the host
SCFA producers Promotes the efficacy of anti-cancer Chaput et al. (2017);
therapies in metastatic Park et al. (2018);
melanoma patients Gopalakrishnan
et al. (2018)
Clostridiaceae Clostridium butyricum 3.88 Mbp; 28.2% Gram-positive, obligate anaerobe, Inhibition of C. difficile in vitro using human Woo et al. (2011)
endospore-former, butyric acid-producing bladder cancer 253J-BV cells
Improved efficacy of H. pylori eradication Shimbo et al. (2005)
therapy in patients
Cholesterol-lowering effect in murine model Kobashi et al. (1983);
Takeda et al. (1983)
Anti-cancer effect in vitro Shinnoh et al. (2013)
Improves efficacy and reduce the Tian et al. (2019)
chemotherapy-induced issues in patients
with lung cancer
Eubacteriaceae Eubacterium limosum 4.15 Mbp; 47.4% Gram-positive, non-spore former, obligate Ameliorative effect on colitis in vitro using Kanauchi et al. (2006)
anaerobe, bacilli T84 cells and in DSS-induced colitis
mice model
Eubacterium hallii 3.51 Mbp; 38.6% Anti-obesity and anti-diabetic effect in db/db Udayappan
mice model et al. (2016)
Christensenellaceae Christensenella minuta 2.56 Mbp; 51.3% Gram-Positive, obligate anaerobe, rod Ameliorative effect on obesity and Goodrich et al.
shaped, secondary bile acid metabolism, associated metabolic disorders in (2014, 2016)
catalase-negative, oxidase negative and mice model
urease negative
Enterococcaceae Enterococcus hirae 2.84 Mbp; 36.72% Gram-positive, facultative anaerobe, non- Improves efficacy of PD-1-based Routy et al. (2018)
spore former, alpha-haemolytic, or immunotherapy against advanced
non-haemolytic melanoma, non-small cell lung cancer
(NSCLC), and renal cell carcinoma (RCC) in
antibiotic treated mice model as well as
in patients with advanced NSCLC, RCC, or

urothelial carcinoma
5
(continued)
6
Table 2. Continued.
Family Genus and species Genome size; GC content Key features Effects studied References
Enhanced efficacy of anti-cancer Daillere et al. (2016)
immunomodulatory agent
cyclophosphamide (CTX) against advanced
lung and ovarian cancer in antibiotic-
treated mice model
Lactobacillaceae Pediococcus 1.75 Mbp; 37.3% Gram-positive, facultative anaerobe, non- Cholesterol-lowering effect in vitro Syakila et al. (2019)
pentosaceus motile, non-spore-former
Phylum: Bacteroidetes
Bacteroidaceae Bacteroides fragilis (PSA 5.1 Mbp; 43.1% Gram-negative, obligate anaerobe, rod- Immunoregulatory functions in vitro Deng et al. (2016)
producing strains) shaped, degradation of the Immunoregulatory functions in germ- Troy and Kasper (2010)
isoflavone genistein free mice
T. P. SINGH AND B. H. NATRAJ
Viral encephalitis in mice model Ramakrishna

et al. (2019)
Bacteroides uniformis 5.16 Mbp; 46.7% Anti-obesity, anti-diabetic and Gauffin et al. (2012)
immunomodulatory effect in high fat
diet-fed mice
Bacteroides vulgatus 4.78 Mbp; 42% Anti-diabetic effect in mice Pedersen et al. (2016)
Bacteroides 4.78 Mbp; 42% Anti-cancer in humans Ulsemer et al. (2016)
xylanisolvens
Bacteroides ovatus 4.78 Mbp; 42% Anti-cancer effect in mice model Ulsemer et al. (2013)
Bacteroides dorei 5.31 Mbp; 41.8% Cholesterol-lowering effect in vitro Gerard et al. (2007)
Bacteroides acidifaciens 4.84 Mbp; 42% Promotes IgA production in intestine of Yanagibashi
gnotobiotic mice model et al. (2013)
Prevotellaceae Prevotella copri 3.92 Mbp; 45% Gram-negative, obligate anaerobe, non- Anti-diabetic effect in mice De Vadder et al. (2016)
spore former
Tannerellaceae Parabacteroides 6.85 Mbp; 43.46% Gram-negative, obligate anaerobe, non-spore Anti-obesity, anti-inflammatory and insulin- Wu et al. (2019)
goldsteinii former, non-motile sensitizing effects in T2 diabetes in the
high fat diet-fed mice
Phylum: Verrucomicrobia
Akkermansiaceae Akkermansia 2.66 Mbp; 55.8% Gram-negative, obligate anaerobe, non- Ameliorative effect against metabolic Barcena et al. (2019)
muciniphila motile, non-spore former, oval-shaped, syndromes and auto-immune diseases in
mucin-degrading bacterium progeroid mice model
Anti-obesity effect, anti-diabetic effect and Ansaldo et al. (2019)
enhanced efficacy of anticancer
immunotherapy such as anti-PD-1
treatment in mice model
Amelioration of amyotrophic lateral sclerosis Blacher et al. (2019)
in mice
Reverse the atherosclerotic lesions, improve Li et al. (2016)
metabolic endotoxemia-induced
inflammation, and restore the gut barrier
in Apoe–/– mice
Phylum: Proteobacteria
Burkholderiaceae Burkholderia cepacia 6.41 Mbp; 66.99% Gram-negative, catalase positive, lactose Improves efficacy of anticancer Pitt et al. (2017)
non-fermenter immunotherapy of CTLA4 blockade in
antibiotic-treated mice
groups are mainly targeted for NGPs candidates. NGP candidate which might be explored further (Wu
Bacteroides fragilis strain ZY-312 (phylum: et al. 2019).
Bacteroidetes), an isolate from breastfed infant faeces, In phylum Firmicutes, the genera mainly
promoted the production of microbicidal molecules Faecalibacterium, Clostridium, Eubacterium, and
and phagocytic functions when co-cultured with colo- Christensenella have been investigated for putative
nocytes and macrophages (Deng et al. 2016). NGPs. F. prausnitzii represents around 5% of the total
Polysaccharide A (PSA) is found as an effector compo- faecal microbiota in healthy adults (Miquel et al. 2013).
nent, produced by particular strains of Bact. fragilis, Several reports have mentioned the decrement of this
which possess immunoregulatory functions (Troy and species in patients suffering from intestinal and meta-
Kasper 2010) and may help prevent viral encephalitis bolic disorders (Martın et al. 2017). Also, some research-
(Ramakrishna et al. 2019) and ameliorate inflammation- ers have claimed this species as a biomarker of choice
related diseases (Lukiw 2016). In contrast, some of the to assist in ulcerative colitis (UC) and Crohn’s disease
Bact. fragilis strains produce fragilysin and enterocins (CD) condition (Lopez-Siles et al. 2017). Researchers
which have been implicated as a risk factor for develop- have illustrated the role of F. prausnitzii in maintaining
ing disease conditions and obviously could not be con- the intestinal homeostasis and integrity owing to its
sidered as a desirable trait in NGP. Similarly, another ability to produce SCFAs, especially butyric acid
PSA producing human faecal isolate, Bact. xylanisolvens (Duncan et al. 2002; Wrzosek et al. 2013). F. prausnitzii
DSM 23964, has been shown to be tolerated in phase 1 has also been reported to induce the proliferation of
human trials (Ulsemer et al. 2012). Also, that heat-inacti- regulatory T-cells associated with IBD (Sedwick 2014).
The genus Faecalibacterium showed the positive influ-
vated preparation of this organism was able to elevate
ence on the efficacy of immune checkpoint blockade
the levels of Thomsen–Friedenreich-specific immuno-
(ICB) therapy used for tumour amelioration which is evi-
globulin M antibodies (anti-TFa IgM) which is believed
dent from the increased CD8þ T-cell infiltration within
to promote a more robust response to cancer (Ulsemer
the tumour environment in addition to the enhanced
et al. 2016) though this study contradicts one of the
frequency of effector CD4þ and CD8þ T-cells in the
defining characteristics of probiotics; that organism
periphery (Chaput et al. 2017; Gopalakrishnan et al.
must be viable. Similarly, the administration of Bact.
2018; Park et al. 2018). Such findings have highlighted
acidifaciens and Bact. ovatus strains has been reported
the tremendous potential of this bacterium both as a
to increase levels of IgA; and, anti-TFNa, IgM, and IgG
therapeutic agent and a prognostic marker in cancer
antibodies in gnotobiotic mice, respectively (Ulsemer
patients. Several Clostridial species (Clostridium acetobu-
et al. 2013; Yanagibashi et al. 2013). In the context of
tylicum, C. beijerinckii, C. butyricum, C. cellulolyticum, C.
the cholesterol–cardiovascular-disease axis, Bact. dorei
ljungdahlii, and C. thermocellum) have presented their
D8 was found to convert cholesterol to coprostanol potential as the NGPs. Researchers have exploited C.
in vitro (Gerard et al. 2007). In the context of obesity- butyricum MIYAIRI 588 for the treatment of C. difficile
related metabolic diseases, the oral administration of infections (Woo et al. 2011), Helicobacter pylori infec-
Bact. uniformis CECT 7771 showed anti-obesity, anti-dia- tions (Shimbo et al. 2005), cardio-vascular diseases
betic and immunomodulatory effects in high fat diet- (CVDs) (Kobashi et al. 1983; Takeda et al. 1983), and
fed mice (Gauffin et al. 2012; Yang et al. 2017). Also, cancer (Shinnoh et al. 2013). Some non-toxigenic strains
Bact. vulgatus showed anti-diabetic effect in mice of C. butyricum have also been found to improve the
model (Pedersen et al. 2016). Genus Prevotella, also efficacy and reduce the chemotherapy-induced issues
belongs to phylum Bacteroidetes, is more prevalent in in patients with lung cancer (Tian et al. 2019).
populations that follow the traditional lifestyle and con- Udayappan et al. (2016) observed improved insulin sen-
sume fresh unprocessed food. Reports mention both sitivity and increased energy metabolism upon the
the positive and negative impacts of P. copri on host administration of Eubacterium hallii in obese and dia-
health. De Vadder et al. (2016) highlighted the import- betic db/db mice. Such effect was attributed to the abil-
ance of P. copri in improving glucose homeostasis via ity of E. hallii to produce SCFAs (butyrate and
intestinal gluconeogenesis which was believed to be propionates) upon fermentation of glucose and the fer-
due to the synthesis of branched-chain fatty acids (suc- mentation intermediates (acetate and lactate). While
cinate) from dietary fibre fermentation. Another bacter- others proposed such metabolic versatility, a respon-
ium from the phylum Bacteroidetes, Parabacteroides sible factor for ability of E. hallii to restore the host–gut
goldsteinii, showed anti-obesity, anti-inflammatory, and microbiota homeostasis (Engels et al. 2016) and ameli-
insulin-sensitizing effects in vitro making it a suitable oration of DSS-induced colitis in mice (Kanauchi et al.
2006). Other workers correlated the intestinal abun- Matson et al. (2018) observed the abundance of Bif. lon-
dance of E. limosum with reduced risk of disease relapse gum, C. aerofaciens, and Ent. faecium in stool samples of
and progression after allo-hematopoietic stem cell clinical melanoma ICB patients responding positively to
transplantation (HSCT) treatment in patients with anti-PD-1 antibody therapy. However, more independ-
haematological malignancies (Mulanovich et al. 2016). ent cohort studies are necessary to validate the efficacy
Another study suggested the probiotic effects of of C. aerofaciens with the outcomes of ICB.
Christensenella minuta against obesity and associated Similarly, Burkholderia cepacia (phylum
metabolic disorders as they observed alteration of Proteobacteria) alone or in combination with B. fragilis
obesity-associated microbial patterns in recipient mice significantly improved efficacy and tolerability of CTLA-
(Goodrich et al. 2014). The authors also reported the 4 blockade via the stimulation of TH1 cell immune
abundance of Christensenellaceae in individuals with responses in antibiotic-treated mice (Pitt et al. 2017).
low body mass index (BMI) (Goodrich et al. 2016). The The potential of human faeces originated Gordonibacter
species of genus Enterococcus are noted both as benefi- urolithinfaciens and Gordonibacter pamelaeae, which
cial gut commensals to causative agents of drug-resist- belongs to Eggerthellaceae family of phylum
ant infections (Boehm and Sassoubre 2014). The Actinobacteria, as potential NGP lies in their ability to
Enterococcus strains from non-human origin are already transform ellagic acid (found in walnuts, strawberries,
available commercially as animal probiotics (Hanchi and pomegranates) into urolithins metabolites. The uro-
et al. 2018). The abundance of Ent. hirae was also lithins A and B and isourolithin A are the key metabo-
observed in stool samples of cancer patients respond- lites which possess antiinflammatory, anti-carcinogenic,
ing to PD-1-PD-L1 blockade in comparison with non- cardioprotective, and neuroprotective properties.
responders (Routy et al. 2018). The studies have Therefore, ellagitannin administration could improve
reported the prognostic effects of Ent. hirae ability to health (Selma et al. 2017).
facilitate immunogenic chemotherapy in patients with Akkermansia muciniphila from phylum
non-small cancer lung carcinoma (NSCLC) or ovarian Verrucomicrobia, represents 5% of the gut bacteria and
cancer which resulted from enhanced activity of mem- utilizes mucin as a sole source of carbon, nitrogen, and
ory T-cells (Daillere et al. 2016; Routy et al. 2018). energy. Reduction of A. muciniphila has been pivoted
Another bacterium of phylum Firmicutes, Pediococcus towards the amelioration of metabolic syndromes and
pentosaceus LAB6 also exhibited a cholesterol-lowering auto-immune diseases. Moreover, it has been believed
effect under in vitro conditions (Syakila et al. 2019). that as A. muciniphila possess the ability to modulate
The genus mainly Bifidobacterium, belonging to phy- host metabolism and immunity in a progeroid mouse
lum Actinobacteria, has been evaluated for their poten- model (Barcena et al. 2019). Also, it has been associated
tial as NGP candidates. Oral administration of Bif. breve with effects such as reduced development of obesity
and Bif. longum mixture significantly potentiates the and insulin resistance in mice, enhanced efficacy of
anti-tumour efficacy of anti-PD-L1 immunotherapy in anticancer immunotherapy such as anti-PD-1 treatment
mice model (Sivan et al. 2015). The increased efficacy (Ansaldo et al. 2019) and amelioration of amyotrophic
was attributed to the activation of DCs, leading to lateral sclerosis in mice (Blacher et al. 2019). Moreover,
enhanced CD8þ T-cell priming and accumulation Li et al. (2016) reported that administration of A. mucini-
within the tumour microenvironment (Sivan et al. phila could reverse the atherosclerotic lesions, improve
2015). Bifidobacterium spp. also gained much attention metabolic endotoxemia-induced inflammation, and
on cancer development and therapy against colitis and ultimately, restore the gut barrier in Apoe–/– mice.
colorectal cancer CRC (Kahouli et al. 2013). In another Even though A. muciniphila shows many beneficial
study, the administration of synbiotic (resistant starch effects, however, some other studies in animals
and Bif. lactis) demonstrated ameliorative effects on reported a reverse situation on an increased abundance
CRC development in azoxymethane (AOM)/DSS- of A. muciniphila in the HFD mice (Schneeberger et al.
induced CRC rat model (Le Leu et al. 2010). Similarly, an 2015) and the diseases multiple sclerosis (MS) and
intervention with oligofructose enriched inulin, Bif. Parkinson’s disease (Heintz-Buschart et al. 2018). More
Lactis, and Lb. rhamnosus significantly improved the studies on its ameliorative effects of A. muciniphila
immune function in polypectomized and colon cancer are necessary.
patients (Roller et al. 2007). Rong et al. (2017) also cor- It is worth mentioning here that the NGP includes
related enhanced CD8þ T-cell memory responses with commensals that have no previous history of safe use.
the prognostic effect of Bif. longum and E. hirae mixture Moreover, like conventional probiotics, the beneficial
in patients with HBV-related hepatocellular carcinoma. effects of NGP are also strain specific. As these
commensals may act as opportunistic pathogens under Selection of potential NGPs

particular circumstances, their safety has to be war-
The identification of NGP is not as easy as for conven-
ranted before human applications. Several strains of
tional probiotics as the target organisms may not be
Clostridium, Bacteroidetes, and Enterococcus are known
culturable. The confirmation of the NGP involves differ-
for their ability to produce toxins and have been associ-
ent phases as depicted in Figure 2. First, the identifica-
ated with various infections. Also, studies on NGPs are tion of potential commensal using next-generation
at very initial stages; therefore, there is a need to sequencing (NGS) platforms is based on understanding
explore the identified NGPs for their detailed functional the practical interaction situation between the host and
as well as safety studies.
Figure 2. Illustration of the strategies commonly employed for development of NGPs.

gut microbiota. It is important to consider the quality of has offered a better platform to edit the genome more
the sample and must strictly adhere to well-designed precisely to binate gene expression or provide novel
standard procedures for sample collection, storage, features to enhance host colonization and promote
detailed sequencing, and data interpretation to get human health. Nevertheless, the editing outcomes can
more accurate and applicable outcomes. vary across the strains and methods (Leenay et al. 2018,
The identified microbe needs to be characterized for 2019). Currently, this probiotic engineering strategy has
genus, species, and strain designations. Other informa- been focussed to upgrade the four important proper-
tion like source of microbe, history of the strains, and ties of existing probiotics as depicted in Figure 3.
history of donor health needs to be documented. This Designer probiotics harbouring virulent genes from
is followed by the isolation of potential NGP using a particular pathobiont is one strategy employed to
advanced technologies of culturomics for subsequent curb specific pathogenic infection. For example, Listeria
functional validation studies. The isolates need to be adhesion protein (LAP), a protein responsible for intes-
investigated for physiological characteristics like resist- tinal adhesion of Listeria monocytogenes via interacting
ance to hostile conditions of GIT; the knack to adhere with host Hsp60 subsequently invade epithelial cells
to mucosal lining is also essential (Chua et al. 2019). through the intracellular route using internalin (InlA or
The disease-specific ameliorative effects of the isolate(s) InlB) proteins. Such pathogen-derived LAP was cloned
at the cellular and molecular levels need to be per- and expressed in Lactobacillus paracasei (LP) using
formed both in vitro using cell lines model and in vivo pLP401-T expression vector. Fascinatingly, the recom-
using animal models of either specific pathogens free binant L. paracasei showed superior adhesion ability to
or GF mice as a proof of concept. Considering the Caco-2 cells and subsequently created significant colon-
safety aspects of the potential NGP, genomic analysis ization and internalization resistance against L. monocy-
for the presence of virulence and drug resistance traits togenes via protective, competitive, and displacement
as well as acute toxicity study in both healthy and mode as compared to wild type strain. Besides, the
immunosuppressed mice must be investigated (Saarela recombinant L. paracasei lowered the transepithelial
2019; Tan et al. 2020). Above all these, the unravelling translocation of L. monocytogenes through Caco-2
of the molecular mechanisms involved in host–NGPs monolayers (Koo et al. 2012). Second, targeting GI infec-
interaction is of paramount importance. A thorough tions and toxins was another important area wherein
understanding of the NGPs and their metabolic pat- recombinant probiotics have been evaluated. In this
terns may provide valuable information to design per- regard, recombinant probiotic E. coli (CWG308) harbour-
sonalized treatment strategies (Graspeuntner et al. ing the genes for glycosyltransferase from Neisseria
2019). Lately, the NGP will be validated for its efficacy meningitidis or Campylobacter jejuni for expressing the
and efficiency by phase 1–3 clinical human studies. chimeric lipopolysaccharide (LPS) was capable of neu-
Also, it is worth emphasizing that like conventional pro- tralizing heat-labile enterotoxin of ETEC with high avid-
biotics the beneficial effects of NGP are also strain spe- ity and thus blocking their uptake by host cells and
cific and dosage dependent. thereby preventing disease. Thus, authors concluded
that the probiotic E. coli (CWG308) can be an effective
prophylactic tool to treat ETEC-induced travelers’ diar-
Designer probiotics as NGPs
rhoea (Paton et al. 2005). To date, the receptor mimick-
As previously mentioned, the term NGPs also consider ing probiotics have been targeted to neutralize the
the genetically modified micro-organisms (GMMs) cholera toxins, Shiga toxin (Stx), an AB5 toxin of Stx-
which have been developed either by addition, dele- producing E. coli STEC strains, as well as Shigella dysen-
tion, or overexpression of specific genes to develop a teriae type 1 (Paton et al. 2010).
robust probiotic strain concerning metabolism, techno- The development of mucosal delivery of vaccines for
logical stress tolerance, GIT survivability, and mucosal large-scale herd immunization is indeed a longstanding
directed delivery of therapeutic and/or prophylactic goal of the World Health Organization (WHO) to over-
molecules to confirm one or more health benefits on come infectious diseases with economic, logistical, and
the host and thus leading to the concept of “bio-drug” safety delivery routes (Wells 2011). The oral mucosal
for the prevention or treatment of various diseases (de vaccine has the tremendous potential to exhibit more
Jesus et al. 2019). Hence, using molecular editing tools, generic secretary immunoglobulin A (IgA) responses
the already proven probiotics may further be modified that can neutralize viruses or their toxins and resist the
and explored to design personalized medicine. The colonization of enteric microbes. Probiotics are majorly
recent emergence of CRISPR/Cas9 genome editing tool administered by oral route with the prehistory of long
Figure 3. Avenues for probiotic engineering to develop NGP with specific targeted action.
and safe usage, they are considered as effective muco- enterica, Streptococcus mutans, Streptococcus pneumo-
sal delivery vehicles for antigens, vaccines, and thera- nia, Vibrio parahaemolyticus, Yersinia pseudotuberculosis,
peutic molecules that masked the problem of and Yersinia pestis) have been successfully expressed
delivering functional proteins (Xu et al. 2019). (surface and extracellular) in probiotic Lactobacillus spp.
Moreover, it is a cost-effective approach for the treat- and found to elicit the protective immune response in
ment of mucosal-associated diseases with reduced pos- animals and human trials (LeCureux and Dean 2018;
sible side effects. On the other hand, genetically Palma et al. 2019; Mohseni et al. 2020). More interest-
engineering the probiotic bacteria to express the tar- ingly, few probiotic strains have been specially
geted antigenic part (multiple proteins and other mole- designed to overcome the inflammatory diseases by
cules) to elicit a specific host response is a cloning genes responsible for secreting the anti-inflam-
targeted approach. matory cytokines (IL10, soluble TNF receptor, trefoil fac-
The fate of probiotic bacteria carrying antigenic part tor), superoxide dismutase (Rottiers et al. 2008;
after mucosal delivery is that the DC cell-mediated anti- Watterlot et al. 2010).
gen recognition and processing assisted by M cells in The ineffectiveness of conventional probiotics to
the follicular-associated epithelium uptake antigens treat specific diseases more precisely as a drug has
and present to the underlying immune cells. Such anti- leveraged to alter with their genome to produce a spe-
gen-presenting DC cells migrate to the draining lymph cific molecule that shoots out the targeted disease
nodes (GALT) to trigger the T and B cell response (Wells more effectively and efficiently. In this regard, several
and Mercenier 2008). To date, several viral antigens recombinant probiotics have been constructed and
(porcine parvovirus, porcine rotavirus, infectious bron- proved to be effective to overcome diabetes, cancer,
chitis virus, hepatitis D virus, coronavirus, foot-and- IBD, bacterial vaginosis, etc. For example, the oral
mouth disease virus, etc.) and bacterial antigens administration of LP expressing the Ang-(1-7) with a
(Bacillus anthracis, Borrelia burgdorferi, Clostridium botu- transepithelial carrier showed lowered blood glucose at
linum, Clostridium perfringens, Clostridium tetani, fasted state, improved the intraperitoneal glucose toler-
Escherichia coli (ETEC), Helicobacter pylori, Salmonella ance test, higher plasma insulin level with increases
insulin expression in islets b-cells (Li et al. 2018). viable bacterial cells per unit time. Besides, incorpor-
Lactobacillus reuteri RC-14 engineered to produce anti- ation of such bacteria into food systems without micro-
HIV proteins for blocking the HIV entry into human per- encapsulation, a technique that requires mechanical
ipheral blood mononuclear cells has potentially low- agitation, will be a difficult task as agitation will lead to
ered the sexual transmission of HIV and subsequent the incorporation of the air leading to the development
bacterial vaginosis (Liu et al. 2007). Recombinant E. coli of an unfavourable toxic niche. Further, the production
Nissle 1917 expressing the two anti-tumour proteins, of bacterial biomass as freeze-dried concentrate
viz. P53 and Tum 5 have significantly affected the viabil- demands freeze-drying with the incorporation of poten-
ity of cancer cell lines (HCT-116, HeLa tumour cells) and tial cryoprotective agents under obligate anaerobic
were delivered effectively to the hypoxic tumour region conditions that need microbial quality control steps to
to exhibit anti-tumour effects in xenograft tumour mice monitor other microbial contaminants and viable cell
model (He et al. 2019). Recently, uricase-expressing counts. Furthermore, for effective micro-encapsulation
genetically engineered probiotic E. coli is shown to under anaerobic conditions, low moisture content
decrease uric acid content and higher urease enzyme in demands the freeze-dried bacterial mass to be milled
the hyperuricaemia rat model (Cai et al. 2020). Oral wherein the milled powder should satisfy the powder
administration of recombinant LP expressing ACE-2 characteristics such as wettability, dispersibility, and
served as a live vector for delivery of human ACE2 sinkability. On the other side, the specific nutritional
responsible for reducing the number of acellular capilla- requirements of all the NGPs are yet to be unravelled.
ries, blocked retinal ganglion cell loss, and decreased However, the few well-studied species of NGPs
retinal inflammatory cytokine expression in two mouse demands complex nutritional requirements, which
models of diabetic retinopathy (Verma et al. 2019). required mucin, rumen fluid, haemin broth, etc. at a
Furthermore, the recombinant Lactococcus lactis laboratory scale. Providing such specific growth require-
expressing LZ8 protein shows the ameliorative effect ments is indeed a difficult task at an industrial scale.
on atherosclerosis and non-alcoholic fatty liver disease Hence, further research focussing to develop an eco-
(NAFLD) in the rabbit model by lowering the gene nomically feasible and suitable medium for the cultiva-
expression of IL-1b in the aorta with decreased intima- tion of NGPs at a large scale is of utmost need. These
medial thickness and foam cells in the sub-endothelial series of technological factors pose potential hurdles
space and therefore suggesting that the Lactococcus- for industrialists looking to formulate NGP therapeutics
expressing LZ8 would be a promising medicine for from commensal gut bacteria.
improving both NAFLD and early atherogenesis owing
to its anti-inflammatory effect (Lee et al. 2020). Table 3
Regulatory guidelines for NGP
represents the compiled information related to
designer probiotics and their specific applications. The commercial exploration of probiotics demands
regulatory approval from the apex bodies that vary
across the globe. Although probiotics are majorly mar-
Techno-functional challenges for NGP
keted as dietary food supplements, based on their
development
intended use there have been several categories made
To commercialize the probiotic strains, the techno-func- in different countries. For instance, probiotics are subca-
tional robustness such as calibre to withstand the pro- tegorized under Dietary Supplements/Drugs/Live
cess treatments, enrichment, freeze-drying, product Biotherapeutic agents/Medical food in the USA,
incorporation, and viability controls during product Functional food in Japan/India/China/Malaysia,
shelf-life need careful evaluation to investigate their Biotherapeutic agent in European countries/Belgium/
feasibility. To the best of our knowledge, several trad- Germany, Dietary food in Italy, Natural health products
itional probiotics of bacterial and yeast domain are cur- in Canada, and Food Supplements in Denmark/
rently been produced at an industrial scale and Sweden/Finland. These countries have developed their
handling of such bacteria strains perhaps remains an regulatory systems to warrant approval of candidate
easy task as they are microaerophilic to aerobic. Of probiotics after assuring safety affairs. For example, the
note, extreme oxygen sensitivity of most putative NGP regulatory authorities like Food for Specified Health Use
candidates significantly hampers the large-scale pro- (FOSHU) in Japan, Joint Health Claims Initiative (JHCI) in
duction. Moreover, several existing industrial plant the UK, Food and Drug Administration (FDA) and
designs and process flow lines cannot notably create an Dietary Supplement Health and Education Act (DSHEA)
anaerobic micro-environment to harness maximum in the USA, European Food Safety Authority (EFSA) in
Table 3. Recombinant probiotics and specific applications.
Recombinant Genes inserted/deleted and
probiotics their source Outcomes Targeted disease Genetic engineering tools used References
E. coli Nissle 1917 Lipase ABC transporter recognition Secretion of human epidermal growth Gastrointestinal Cloning and heat shock transformation Choi et al. (2012)
domain 3 (LARD) and EFG genes factor (EGF) in conjunction with the mucosal injuries
have been cloned into pKK223- lipase ABC transporter recognition
3 vector domain (LARD) for proliferative re-
epithelialization
Lb. plantarum 256 Der p 1 of the house dust mite was Inhibition of IFN-gamma and IL-5 Vaccine against allergy Cloning and transformation by Kruisselbrink
cloned into pLP503 vector production in mice model electroporation et al. (2001)
L. lactis NZ9000 beta-lactoglobulin gene was expressed Mice showed increased tolerance Milk allergy Not disclosed Adel-Patient
in pCYT expression vector towards BLG upon immunization. et al. (2005)
Moreover, strains showed
development of a Th1 response
inhibiting the Th2, significant
decrease in IgE response
L. lactis MG1363 Tumour necrosis factor related Viability of HCT-116 and SW480 Cancer Cloning and electroporation Bohlul et al. (2019)
apoptosis inducing ligand (TRAIL) cancerous cell line was significantly
was cloned and expressed in lowered besides change in the
pNZ7021 vector expression of Bax and Bcl2 genes
(apoptosis genes).
L. lactis Cloning nucleotides coding IL-8 and Expression and secretion of IL-25 and Inflammatory Cloning and electroporation Langella et al. (2019)
TSLP under the control of a thymic stromal lymphopoietin bowel disease
promoter GroESL in in vitro (TSLP) in biologically active form
synthesized plasmid that diminished inflammation in
dextran sulphate sodium (DSS) and
dinitrobenzene sulphonic acid
(DNBS) induced inflammatory colitis
mice model.
E. coli Nissle 1917 Genes coding for F4 and F18 fimbriae) Recombinant strain showed adherence ETEC Transformation of pCas and pTarget Ou et al. (2020)
from enterotoxigenic E. coli (ETEC) to porcine intestinal epithelial cell associated diarrhoea plasmid by electroporation followed
have been cloned in an lines. The serum antibodies from by CRISPR-Cas9 mediated
expression vector the recombinant E. coli Nissle 1917 genome editing
immunized mice and piglets
significantly inhibited the
adherence of F4þ or both F4þ and
F18þ ETEC wildtype strains to
porcine intestinal cell lines in vitro.
Immunization of mice also resulted
in the development of anti-F4, and
both anti-F4 and anti-F18 IgG
immune response.
L. lactis NZ9000 Genes for expressing dendritic cell- Chickens immunized with recombinant Vaccine against Cloning and electrotransformation Li et al. (2020)
targeting peptide (DCpep) and E. L. lactis NZ9000 showed higher Eimeriatenella
tenella 3-1E protein from levels of 3-1E specific serum IgG infection
Eimeriatenella were cloned into and secretory IgA (sIgA) in caecal
pTX8048 expression vector and lavage, higher proportions of CD4þ
transformed to lactococci and CD8aþ cells in peripheral
blood, and higher mRNA expression
levels of IL-2 and IFN-c in
the spleen.
(continued)
13
14
Table 3. Continued.
Recombinant Genes inserted/deleted and
probiotics their source Outcomes Targeted disease Genetic engineering tools used References
Lb. acidophilus Cloning the genes encoding MPER-specific IgA secretion in mice. Vaccine against HIV Cloning and electrotransformation Abdo et al. (2019)
membrane-proximal external region
(MPER) from HIV, MPER þ IL-1B
adjuvant, MPER þ salmonella FliC
adjuvant in an expression vector
Lb. gasseri ATCC 33323 Cloning specific gene expressing the Diabetic rats fed GLP-1-secreting Diabetes Cloning and transformation Duan et al. (2015)
GLP 1(1-37) into pBluescript bacteria showed significant
expression vector increases in insulin levels and, were
significantly more glucose tolerant.
The rats developed insulin-
T. P. SINGH AND B. H. NATRAJ
producing cells within the upper

intestine in numbers sufficient to
replace 25–33% of the insulin
capacity of non-diabetic
healthy rats.
Lb. plantarum and HIV-1 fusion inhibitors (FI-1. FI-2, and Viral infectivity of primary HIV-1 HIV Cloning and transformation Pusch et al. (2006)
Lb. gasseri FI-3) were introduced into the isolates and SHIV-162p3 was
previously developed shuttle neutralized by up to 98% and 72%,
vector pTSV2 respectively, by 10% (v/v)
lactobacillus supernatant containing
fusion inhibitor FI-3.
L. lactis subsp. cremoris Cloning GLP-1 (1–37) genes into MG1363 administration significantly Alzheimer’s disease Cloning and transformation Chen et al. (2018)
(strain MG1363) pMG36e and pLIVE vector restored the spatial learning and
memory impairment of mice
caused by LPS. There was
suppressed glia activation and
amyloid-b accumulation, and down-
regulated inflammatory expressions
of COX-2, TLR-4, TNF-a, and IL-1b.
Lb. casei 393 Cloning a, e, b1, and b2 toxoids Increase in the antigen-specific Vaccine against Cloning and electrotransformation Ding et al. (2019)
genes from Clostridium perfringens secretory IgA (sIgA)-based mucosal Clostridium
using an expression vector and IgG-based humoral immune perfringens
responses. High levels (p< 0.05) of
cytokines IL-2, IL-4, IL-10, IL-12, IL-
17, and IFN-c were produced in
immunized mice.
Lb. casei OMP-19 Gene encoding OMP19 antigen from Lb. casei based vaccine represented a Oral vaccine against Cloning and electrotransformation Mohammadi and
Brucella abortus was primarily very good general and mucosal Brucella abortus Golchin (2020)
amplified and cloned into an immune responses protective
expression vector pT1NX against challenges with virulent B.
abortus 544 strain compared with
negative control recipient groups.
L. lactis NZ9000 Cloning the TNF-a-binding antibodies Recombinant L. lactis at 1 108 CFU/ IBD Cloning and electrotransformation Simcic et al. (2019)
mL bound as much as 62.8%
(p ¼ 0.026) of TNF-a in IBD-mucosa
supernatants compared with the
control strain.
(continued)
Europe, State Food and Drug Administration (SFDA) in

China, Food Safety and Standards Authority of India
Bohlul et al. (2019)

Zeng et al. (2020)
References (FSSAI) in India, Canadian Food and Drugs Act under
Natural Health Products Regulations in Canada, Food
Safety and Quality Division (FSQD), Drug Control
Authority, the National Pharmaceutical Control Bureau
(NPCB) in Malaysia, and National Health Surveillance
Agency in Brazil have been recognized worldwide.
Genetic engineering tools used
Overall, the joint expert consultants of FAO/WHO co-

Cloning and electrotransformation
ordinate in assuring the health and nutritional proper-
Cloning and electroporation

ties of probiotics as per scientific recommendations
Cloning and transfection
(Arora and Baldi 2015).

As EFSA is the responsible regulatory agency in the
European Union, EFSA Scientific Committee provides a
generic concept on qualified presumption of safety
(QPS) status to the microorganisms that are intended to
food and feed applications. Under which, the number
of beneficial microbes accorded QPS status has been
Anti-parasitic infections
recently revisited and the list does not contain any gut-
hyperpermeability
Targeted disease
derived NGPs or engineered probiotics except the gen-

era bifidobacteria (Koutsoumanis et al. 2019). However,
the biological medicinal products of active substances
Intestinal
Cancer
derived from bacterium or yeast need authorization

and approval from the European Medicines Agency.
Furthermore, the Biosimilar Medicinal Products Working
increased significantly in mice orally
integrity. Reduced the expression of

immune response was up-regulated
transcription factor-kappa B (NF-jB)

inflammatory cell infiltration, down-
inflammation-related molecules and
Recombinant L. lactis producing TRAIL

glandular structure destruction and
inoculated with NC8-pSIP409-pgsA-
p65 and its inhibitor IjBa in mice.
Party (BMWP) recommended the Committee for

human colon adenocarcinoma cell
protein can induce apoptosis in
Levels of serum IgG1 and mucosal
regulated expression of several

mIL-4, and the Th2 phenotype
secretory IgA (sIgA) were both
Medicinal Products for Human Use (CHMP) for the regu-

Significant reduction in colonic
preserved epithelial barrier
latory assessment of biosimilar medicinal products

lines SW480 and HCT116.
phosphorylated nuclear
(https://www.ema.europa.eu/en/human-regulatory/rese
Outcomes
arch-development/innovation-medicines; https://www.
ebe-biopharma.eu/biotherapeutics/). Likewise, the
European Directorate for the Quality of Medicines
in mice.
(EDQM) has framed a Live Biotherapeutic Products

Working Party in 2014 to develop a monograph for
LBPs to harmonize quality standards for LBPs as bio-
logical medicinal products (https://www.edqm.eu/en/
forms of human TRAIL cloned into
Recombinant plasmid was designed
biotherapeutics).
mHD-5 was cloned into L. lactis
The secreted or cell wall-anchored

using murine IL-4 co-expressed
Genes inserted/deleted and
Gene coding for human defensin
In the USA, live microbes fall under the category of

NZ9000SHD-5 via pMD19-T
pNZ7021 expression vector

with pgsA anchor system
foods or dietary supplements when used as foods, or

their source
LBP agents when referring to use as a drug. In this con-

expression vector
text, based on disease-specific use of commensal

microbes isolated from the human host (NGPs), they
are classified under LBPs. Besides, the NGPs of engi-
neered construct or designer probiotics fall under the
category of recombinant LBPs. The recombinant LBPs
are the LBPs composed of microorganisms that have
Table 3. Continued.
been genetically modified through the purposeful add-

L. lactis NZ9000SHD-5
ition, deletion, or modification of genetic material (US

Lb. plantarum NC8
Food and Drug Administration 2016). Furthermore, the

Recombinant
Centre for Biologics Evaluation and Research (CBER) of

probiotics
L. lactis
the FDA regulates the LBP section. CBER demands strin-

gent in vitro characterization of probiotics both at
phenotypic and genotypic levels including identity, References

antimicrobial resistance, mutations, virulence factors,
Abdo Z, LeCureux J, LaVoy A, Eklund B, Ryan EP, Dean GA.
and mode of action in disease mitigation. It is worth 2019. Impact of oral probiotic Lactobacillus acidophilus
noting that LBPs should be produced by good manu- vaccine strains on the immune response and gut micro-
facturing practice (GMP) standards like vaccines. For biome of mice. PLoS One. 14(12):e0225842.
successful marketing approval, LBPs should undergo Adel-Patient K, Ah-Leung S, Creminon C, Nouaille S, Chatel
JM, Langella P, Wal JM. 2005. Oral administration of
well designed preclinical and clinical trials to evaluate recombinant Lactococcus lactis expressing bovine beta-
the safety, efficacy, and effectiveness of probiotics (Ross lactoglobulin partially prevents mice from sensitization.
et al. 2008). Clin Exp Allergy. 35(4):539–546.
Ansaldo E, Slayden LC, Ching KL, Koch MA, Wolf NK, Plichta
DR, Brown EM, Graham DB, Xavier RJ, Moon JJ, et al. 2019.
Conclusions Akkermansia muciniphila induces intestinal adaptive
immune responses during homeostasis. Science.
Conventional probiotics are generic in action and 364(6446):1179–1184.
restricted to a limited range of organisms. Arora M, Baldi A. 2015. Regulatory categories of probiotics
Understanding gut microbiota and its unique relation- across the globe: a review representing existing and rec-
ommended categorization. Indian J Med Microbiol. 33(5):
ship with the host health state has changed the para-
2–10.
digm and has widened the scope to explore a broad Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T,
range of organisms over the phylogenetic stretch. In- Mende DR, Fernandes GR, Tap J, Bruls T, Batto JM, et al.
depth scientific studies on understanding health bene- 2011. Enterotypes of the human gut microbiome. Nature.
fits associated with particular commensal may lead to 473(7346):174–180.
Barcena C, Valdes-Mas R, Mayoral P, Garabaya C, Durand S,
the discovery of novel microbes which may further be Rodrıguez F, Fernandez-Garcıa MT, Salazar N, Nogacka AM,
explored for designing disease specific personalized Garatachea N, et al. 2019. Healthspan and lifespan exten-
medicine. Eventually, the studies may find the unidenti- sion by fecal microbiota transplantation into progeroid
fied microorganisms with unusual properties and some mice. Nat Med. 25(8):1234–1242.
Barzegari A, Saei AA. 2012. Designing probiotics with respect
may belong to well-known pathogenic groups that pre-
to the native microbiome. Future Microbiol. 7(5):571–575.
sent difficulties for research, industrial exploitation, and Blacher E, Bashiardes S, Shapiro H, Rothschild D, Mor U, Dori-
regulatory agencies. Such health promoting non-trad- Bachash M, Kleimeyer C, Moresi C, Harnik Y, Zur M, et al.
itional microorganisms are generally referred to as 2019. Potential roles of gut microbiome and metabolites
NGPs; which itself is regulated under the LBP guidelines in modulating ALS in mice. Nature. 572(7770):474–480.
Boehm AB, Sassoubre LM. 2014. Enterococci as indicators of
of FDA if used for pharmaceutical applications. environmental fecal contamination. In: Glimore MS,
Identification of NGPs is most challenging as it is com- Clewell DB, Ike Y, editors. Enterococci: from commensals
pletely an assumption-based approach and mainly to leading cause of drug resistant infection. Boston:
based on correlation studies comparing relative abun- Massachusetts Eye and Ear Infirmary.
Bohlul E, Hasanlou F, Taromchi AH, Nadri S. 2019. TRAIL-
dance levels of microorganisms among health and dis-
expressing recombinant Lactococcus lactis induces apop-
eased subjects. Once identified, the organism must be tosis in human colon adenocarcinoma SW480 and HCT116
validated for various functionality, safety and techno- cells. J Appl Microbiol. 126(5):1558–1567.
logical parameters using appropriate in silico, in vitro, Cai L, Li Q, Deng Y, Liu X, Du W, Jiang X. 2020. Construction
in vivo models. The uncommon growth requirements and expression of recombinant uricase-expressing genetic-
ally engineered bacteria and its application in rat model
need to be considered to achieve large-scale produc-
of hyperuricemia. Int J Mol Med. 45(5):1488–1500.
tion of LBP and formulation must ensure the viability Chaput N, Lepage P, Coutzac C, Soularue E, Le Roux K,
and bioactivity of the culture until consumed. The Monot C, Boselli L, Routier E, Cassard L, Collins M, et al.
developed product must be examined for safety, effi- 2017. Baseline gut microbiota predicts clinical response
cacy, and efficiency and dose confirmation by phase and colitis in metastatic melanoma patients treated with
ipilimumab. Ann Oncol. 28(6):1368–1379.
1–3 clinical trials and must pass the regulatory approval Chen T, Tian P, Huang Z, Zhao X, Wang H, Xia C, Wang L,
hurdles. Conclusively, the concept of NGP is at fledgling Wei H. 2018. Engineered commensal bacteria prevent sys-
stage but is highly promising and requires extensive temic inflammation-induced memory impairment and
research for its exploitation as preventive medicine. amyloidogenesis via producing GLP-1. Appl Microbiol
Biotechnol. 102(17):7565–7575.
Choi HJ, Ahn JH, Park SH, Do KH, Kim J, Moon Y. 2012.
Enhanced wound healing by recombinant Escherichia coli
Disclosure statement
Nissle 1917 via human epidermal growth factor receptor
The authors declare that there is no conflict of interest. in human intestinal epithelial cells: therapeutic implication
using recombinant probiotics. Infect Immun. 80(3): Goodrich JK, Waters JL, Poole AC, Sutter JL, Koren O,
1079–1087. Blekhman R, Beaumont M, Van Treuren W, Knight R, Bell
Chua JCL, Hale JDF, Silcock P, Bremer PJ. 2019. Bacterial sur- JT, et al. 2014. Human genetics shape the gut microbiome.
vival and adhesion for formulating new oral probiotic Cell. 159(4):789–799.
foods. Crit Rev Food Sci Nutr. 60(17):1–12. Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews
Daillere R, Vetizou M, Waldschmitt N, Yamazaki T, Isnard C, MC, Karpinets TV, Prieto PA, Vicente D, Hoffman K, Wei SC,
Poirier-Colame V, Duong CPM, Flament C, Lepage P, et al. 2018. Gut microbiome modulates response to anti-
Roberti MP, et al. 2016. Enterococcus hirae and Barnesiella PD-1 immunotherapy in melanoma patients. Science.
intestinihominis facilitate cyclophosphamide-induced thera- 359(6371):97–103.
peutic immunomodulatory effects. Immunity. 45(4): Graspeuntner S, Waschina S, K€ unzel S, Twisselmann N,
931–943. Rausch TK, Cloppenborg-Schmidt K, Zimmermann J,
de Jesus LCL, Lima FA, Coelho-Rocha ND, da Silva TF, Paz J, Viemann D, Herting E, Go €pel W, et al. 2019. Gut dysbiosis
Azevedo V, Mancha-Agresti P, Drumond MM. 2019. with bacilli dominance and accumulation of fermentation
Recombinant probiotics and microbiota modulation as a products precedes late-onset sepsis in preterm infants.
good therapy for diseases related to the GIT. In: Salanta Clin Infect Dis. 69(2):268–277.
LC, editor. The health benefits of foods—current know- Hanchi H, Mottawea W, Sebei K, Hammami R. 2018. The
ledge and further development. London: IntechOpen genus Enterococcus: between probiotic potential and
Limited. DOI:10.5772/intechopen.88325 safety concerns—an update. Front Microbiol. 9:1791.
De Vadder F, Kovatcheva-Datchary P, Zitoun C, Duchampt A, He L, Yang H, Tang J, Liu Z, Chen Y, Lu B, He H, Tang S, Sun
Backhed F, Mithieux G. 2016. Microbiota-produced succin- Y, Liu F, et al. 2019. Intestinal probiotics E. coli Nissle 1917
ate improves glucose homeostasis via intestinal gluconeo- as a targeted vehicle for delivery of p53 and Tum-5 to
genesis. Cell Metab. 24(1):151–157. solid tumors for cancer therapy. J Biol Eng. 13(1):58.
Deng H, Li Z, Tan Y, Guo Z, Liu Y, Wang Y, Yuan Y, Yang R, Heintz-Buschart A, Pandey U, Wicke T, Sixel-Do €ring F, Janzen
Bi Y, Bai Y, et al. 2016. A novel strain of Bacteroides fragilis A, Sittig-Wiegand E, Trenkwalder C, Oertel WH,
enhances phagocytosis and polarises M1 macrophages. Sci Mollenhauer B, Wilmes P. 2018. The nasal and gut micro-
Rep. 6:29401.
biome in Parkinson’s disease and idiopathic rapid eye
Ding G, Bai J, Feng B, Wang L, Qiao X, Zhou H, Jiang Y, Cui
movement sleep behavior disorder. Mov Disord. 33(1):
W, Tang L, Li Y, et al. 2019. An EGFP-marked recombinant
88–98.
lactobacillus oral tetravalent vaccine constitutively express-
Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B,
ing a, e, b1, and b2 toxoids for Clostridium perfringens elic-
Morelli L, Canani RB, Flint HJ, Salminen S, et al. 2014.
its effective anti-toxins protective immunity. Virulence.
Expert consensus document. The International Scientific
10(1):754–767.
Association for Probiotics and Prebiotics consensus state-
Duan FF, Liu JH, March JC. 2015. Engineered commensal bac-
ment on the scope and appropriate use of the term pro-
teria reprogram intestinal cells into glucose-responsive
biotic. Nat Rev Gastroenterol Hepatol. 11(8):506–514.
insulin-secreting cells for the treatment of diabetes.
Kahouli I, Tomaro-Duchesneau C, Prakash S. 2013. Probiotics
Diabetes. 64(5):1794–1803.
Duncan SH, Hold GL, Harmsen HJ, Stewart CS, Flint HJ. 2002. in colorectal cancer (CRC) with emphasis on mechanisms
Growth requirements and fermentation products of of action and current perspectives. J Med Microbiol. 62(Pt
Fusobacterium prausnitzii, and a proposal to reclassify it as 8):1107–1123.
Faecalibacterium prausnitzii gen. nov., comb. nov. Int J Syst Kanauchi O, Fukuda M, Matsumoto Y, Ishii S, Ozawa T,
Evol Microbiol. 52:2141–2146. Shimizu M, Mitsuyama K, Andoh A. 2006. Eubacterium
Engels C, Ruscheweyh HJ, Beerenwinkel N, Lacroix C, Schwab limosum ameliorates experimental colitis and metabolite
C. 2016. The common gut microbe Eubacterium hallii also of microbe attenuates colonic inflammatory action with
contributes to intestinal propionate formation. Front increase of mucosal integrity. World J Gastroenterol. 12(7):
Microbiol. 7:713. 1071–1077.
Feng Q, Chen WD, Wang YD. 2018. Gut microbiota: an inte- Kho ZY, Lal SK. 2018. The human gut microbiome – a poten-
gral moderator in health and disease. Front Microbiol. 9: tial controller of wellness and disease. Front Microbiol. 9:
151. 1835.
Fuller R. 1989. Probiotics in man and animals. J Appl Kobashi K, Takeda Y, Itoh H, Hase J. 1983. Cholesterol-lower-
Bacteriol. 66(5):365–378. ing effect of Clostridium butyricum in cholesterol-fed rats.
Gauffin CP, Santacruz A, Moya A, Sanz Y. 2012. Bacteroides Digestion. 26(4):173–178.
uniformis CECT 7771 ameliorates metabolic and immuno- Kollath W. 1953. The increase of the diseases of civilization
logical dysfunction in mice with high-fat-diet induced and their prevention. Munch Med Wochenschr. 95(47):
obesity. PLoS One. 7(7):e41079. 1260–1262.
Gerard P, Lepercq P, Leclerc M, Gavini F, Raibaud P, Juste C. Koo OK, Amalaradjou MAR, Bhunia AK. 2012. Recombinant
2007. Bacteroides sp. strain D8, the first cholesterol-reduc- probiotic expressing Listeria adhesion protein attenuates
ing bacterium isolated from human feces. Appl Environ Listeria monocytogenes virulence in vitro. PLoS One. 7(1):
Microbiol. 73(18):5742–5749. e29277.
Goodrich JK, Davenport ER, Beaumont M, Jackson MA,
Koutsoumanis K, Allende A, Alvarez-Ord n
o ~ez A, Bolton D,
Knight R, Ober C, Spector TD, Bell JT, Clark AG, Ley RE. Bover-Cid S, Chemaly M, Davies R, De Cesare A, Hilbert F,
2016. Genetic determinants of the gut microbiome in UK Lindqvist R, et al. 2019. Update of the list of QPS-recom-
twins. Cell Host Microbe. 19(5):731–743. mended biological agents intentionally added to food or
feed as notified to EFSA 10: suitability of taxonomic units associated with anti-PD-1 efficacy in metastatic melanoma
notified to EFSA until March 2019. EFSA J. 17(7):5753. patients. Science. 359(6371):104–108.
Kruisselbrink A, Heijne Den Bak-Glashouwer MJ, Havenith Miquel S, Martin R, Rossi O, Bermudez-Humaran LG, Chatel
CEG, Thole JER, Janssen R. 2001. Recombinant JM, Sokol H, Thomas M, Wells JM, Langella P. 2013.
Lactobacillus plantarum inhibits house dust mite-specific T- Faecalibacterium prausnitzii and human intestinal health.
cell responses. Clin Exp Immunol. 126(1):2–8. Curr Opin Microbiol. 16(3):255–261.
Langella P, Chatel JM, Bermudez-Humaran L, Aubry C, Goffin Mohammadi E, Golchin M. 2020. High protection of mice
L, Favre-Zimmerli S, Chvatchko Y. 2019. Lactococcus lactis against Brucella abortus by oral immunization with recom-
producing TSLP or IL-25 and their uses as probiotics and binant probiotic Lactobacillus casei vector vaccine, express-
therapeutics. United States patent US 10,238,697. ing the outer membrane protein OMP19 of Brucella
Le Leu RK, Hu Y, Brown IL, Woodman RJ, Young GP. 2010. species. Comp Immunol Microbiol Infect Dis. 70:101470.
Synbiotic intervention of Bifidobacterium lactis and resist- Mohseni AH, Taghinezhad SS, Keyvani H. 2020. The first clin-
ant starch protects against colorectal cancer development ical use of a recombinant Lactococcus lactis expressing
in rats. Carcinogenesis. 31(2):246–251. human papillomavirus type 16 E7 oncogene oral vaccine:
LeCureux JS, Dean GA. 2018. Lactobacillus mucosal vaccine a phase I safety and immunogenicity trial in healthy
vectors: immune responses against bacterial and viral anti- women volunteers. Mol Cancer Ther. 19(2):717–727.
gens. mSphere. 3(3):e00061-18. Mulanovich VE, Desai PA, Popat UR. 2016. Allogeneic stem
Lee MF, Chiang CH, Lin SJ, Song PP, Liu HC, Wu TJ, Lin WW. cell transplantation for HIV-positive patients with hemato-
2020. Recombinant Lactococcus lactis expressing ling zhi 8 logic malignancies. AIDS. 30(17):2653–2657.
protein ameliorates nonalcoholic fatty liver and early Okumura R, Takeda K. 2018. Maintenance of intestinal
atherogenesis in cholesterol-fed rabbits. Biomed Res Int. homeostasis by mucosal barriers. Inflamm Regen. 38:5.
2020:3495682. O’Toole PW, Marchesi JR, Hill C. 2017. Next-generation probi-
Leenay RT, Vento JM, Shah M, Martino ME, Leulier F, Beisel otics: the spectrum from probiotics to live biotherapeutics.
CL. 2018. Streamlined, recombinase-free genome editing Nat Microbiol. 2:17057.
with CRISPR-Cas9 in Lactobacillus plantarum reveals bar- Ou B, Jiang B, Jin D, Yang Y, Zhang M, Zhang D, Zhao H, Xu
riers to efficient editing. bioRxiv, 352039. M, Song H, Wu W, et al. 2020. Engineered recombinant
Leenay RT, Vento JM, Shah M, Martino ME, Leulier F, Beisel Escherichia coli probiotic strains integrated with F4 and
CL. 2019. Genome editing with CRISPR-Cas9 in F18 fimbriae cluster genes in the chromosome and their
Lactobacillus plantarum revealed that editing outcomes assessment of immunogenic efficacy in vivo. ACS Synth
can vary across strains and between methods. Biotechnol Biol. 9(2):412–426.
J. 14(3):e1700583. Palma ML, Garcia-Bates TM, Martins FS, Douradinha B. 2019.
Li J, Lin S, Vanhoutte PM, Woo CW, Xu A. 2016. Akkermansia Genetically engineered probiotic Saccharomyces cerevisiae
muciniphila protects against atherosclerosis by preventing strains mature human dendritic cells and stimulate Gag-
metabolic endotoxemia-induced inflammation in Apoe–/– specific memory CD8þ T cells ex vivo. Appl Microbiol
mice. Circulation. 133(24):2434–2446. Biotechnol. 103(13):5183–5192.
Li G, Ma C, Wang D, Chen W, Ma D. 2020. Recombinant Park YJ, Kuen DS, Chung Y. 2018. Future prospects of
Lactococcus lactis co-expressing dendritic cell target pep- immune checkpoint blockade in cancer: from response
tide and E. tenella 3-1E protein: immune response and effi- prediction to overcoming resistance. Exp Mol Med. 50(8):
cacy against homologous challenge. Food Agric Immunol. 1–13.
31(1):379–392. Paton AW, Jennings MP, Morona R, Wang H, Focareta A,
Liu JJ, Reid G, Jiang Y, Turner MS, Tsai CC. 2007. Activity of Roddam LF, Paton JC. 2005. Recombinant probiotics for
HIV entry and fusion inhibitors expressed by the human treatment and prevention of enterotoxigenic Escherichia
vaginal colonizing probiotic Lactobacillus reuteri RC-14. coli diarrhea. Gastroenterology. 128(5):1219–1228.
Cell Microbiol. 9(1):120–130. Paton AW, Morona R, Paton JC. 2010. Bioengineered bugs
Li Q, Xu K, Du TAO, Zhu P, Verma A. 2018. Recombinant pro- expressing oligosaccharide receptor mimics: toxin-binding
biotics expressing angiotensin-(1-7) improves glucose probiotics for treatment and prevention of enteric infec-
metabolism and diabetes-induced renal and retinal injury. tions. Bioeng Bugs. 1(3):172–177.
Diabetes. 67(Suppl. 1):33-LB. Pedersen HK, Gudmundsdottir V, Nielsen HB, Hyotylainen T,
Lopez-Siles M, Duncan SH, Garcia-Gil LJ, Martinez-Medina M. Nielsen T, Jensen BAH, Forslund K, Hildebrand F, Prifti E,
2017. Faecalibacterium prausnitzii: from microbiology to Falony G, et al. 2016. Human gut microbes impact host
diagnostics and prognostics. ISME J. 11(4):841–852. serum metabolome and insulin sensitivity. Nature.
Lukiw WJ. 2016. Bacteroides fragilis lipopolysaccharide and 535(7612):376–381.
inflammatory signaling in Alzheimer’s disease. Front Petrof EO, Khoruts A. 2014. From stool transplants to next-
Microbiol. 7:1544. generation microbiota therapeutics. Gastroenterology.
Martın R, Miquel S, Benevides L, Bridonneau C, Robert V, 146(6):1573–1582.
Hudault S, Chain F, Berteau O, Azevedo V, Chatel JM, et al. Pitt JM, Vetizou M, Gomperts Boneca I, Lepage P,
2017. Functional characterization of novel Faecalibacterium Chamaillard M, Zitvogel L. 2017. Enhancing the clinical
prausnitzii strains isolated from healthy volunteers: a step coverage and anticancer efficacy of immune checkpoint
forward in the use of F. prausnitzii as a next-generation lockade through manipulation of the gut microbiota.
probiotic. Front Microbiol. 8:1226. OncoImmunology. 6(1):e1132137.
Matson V, Fessler J, Bao R, Chongsuwat T, Zha Y, Alegre ML, Pusch O, Kalyanaraman R, Tucker LD, Wells JM, Ramratnam
Luke JJ, Gajewski TF. 2018. The commensal microbiome is B, Boden D. 2006. An anti-HIV microbicide engineered in
commensal bacteria: secretion of HIV-1 fusion inhibitors MIYAIRI 588 shows antitumor effects by enhancing the
by lactobacilli. AIDS. 20(15):1917–1922. release of TRAIL from neutrophils through MMP-8. Int J
Ramakrishna C, Kujawski M, Chu H, Li L, Mazmanian SK, Oncol. 42(3):903–911.
Cantin EM. 2019. Bacteroides fragilis polysaccharide A indu- Simcic S, Berlec A, Stopinsek S, Strukelj B, Orel R. 2019.
ces IL-10 secreting B and T cells that prevent viral enceph- Engineered and wild-type L. lactis promote anti-inflamma-
alitis. Nat Commun. 10(1):2153. tory cytokine signalling in inflammatory bowel disease
Rinninella E, Raoul P, Cintoni M, Franceschi F, Miggiano GAD, patient’s mucosa. World J Microbiol Biotechnol. 35(3):45.
Gasbarrini A, Mele MC. 2019. What is the healthy gut Sivan A, Corrales L, Hubert N, Williams JB, Aquino-Michaels K,
microbiota composition? A changing ecosystem across Earley ZM, Benyamin FW, Lei YM, Jabri B, Alegre ML, et al.
age, environment, diet, and diseases. Microorganism. 7(1): 2015. Commensal Bifidobacterium promotes antitumor
14. immunity and facilitates anti-PD-L1 efficacy. Science.
Roller M, Clune Y, Collins K, Rechkemmer G, Watzl B. 2007. 350(6264):1084–1089.
Consumption of prebiotic inulin enriched with oligofruc- Syakila RN, Lim SM, Agatonovic-Kustrin S, Lim FT, Ramasamy
tose in combination with the probiotics Lactobacillus K. 2019. In vitro assessment of pediococci- and lactoba-
rhamnosus and Bifidobacterium lactis has minor effects on cilli-induced cholesterol-lowering effect using digitally
selected immune parameters in polypectomised and colon enhanced high-performance thin-layer chromatography
cancer patients. Br J Nutr. 97(4):676–684. and confocal microscopy. Anal Bioanal Chem. 411(6):
Rong Y, Dong Z, Hong Z, Jin Y, Zhang W, Zhang B, Mao W, 1181–1192.
Kong H, Wang C, Yang B, et al. 2017. Reactivity toward Takeda Y, Itoh H, Kobashi K. 1983. Effect of Clostridium butyri-
Bifidobacterium longum and Enterococcus hirae demon- cum on the formation and dissolution of gallstones in
strate robust CD8þ T cell response and better prognosis experimental cholesterol cholelithiasis. Life Sci. 32(5):
in HBV-related hepatocellular carcinoma. Exp Cell Res. 541–546.
358(2):352–359. Tan H, Wang C, Zhang Q, Tang X, Zhao J, Zhang H, Zhai Q,
Ross JJ, Boucher PE, Bhattacharyya SP, Kopecko DJ, Chen W. 2020. Preliminary safety assessment of a new
Sutkowski EM, Rohan PJ, Chandler DK, Vaillancourt J. 2008. Bacteroides fragilis isolate. Food Chem Toxicol. 135:110934.
Considerations in the development of live biotherapeutic Tian Y, Li M, Song W, Jiang R, Li YQ. 2019. Effects of probiot-
products for clinical use. Curr Issues Mol Biol. 10(1–2):
ics on chemotherapy in patients with lung cancer. Oncol
13–16.
Lett. 17(3):2836–2848.
Rottiers P, Vandenbroucke K, Iserentant D. 2008. Use of
Troy EB, Kasper DL. 2010. Beneficial effects of Bacteroides fra-
recombinant yeast strain producing an anti-inflammatory
gilis polysaccharides on the immune system. Front Biosci
compound to treat colitis. United States patent US 12/
(Landmark Ed). 15:25–34.
088,927.
Udayappan S, Manneras-Holm L, Chaplin-Scott A, Belzer C,
Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT,
Herrema H, Dallinga-Thie GM, Duncan SH, Stroes ESG,
Daillere R, Fluckiger A, Messaoudene M, Rauber C, Roberti
Groen AK, Flint HJ, et al. 2016. Oral treatment with
MP, et al. 2018. Gut microbiome influences efficacy of PD-
Eubacterium hallii improves insulin sensitivity in db/db
1-based immunotherapy against epithelial tumors.
mice. NPJ Biofilms Microbiomes. 2:16009.
Science. 359(6371):91–97.
Ulsemer P, Henderson G, Toutounian K, Lo €ffler A, Schmidt J,
Saarela MH. 2019. Safety aspects of next generation probiot-
Karsten U, Blaut M, Goletz S. 2013. Specific humoral
ics. Curr Opin Food Sci. 30:8–13.
Savoia C, Volpe M, Grassi G, Borghi C, Rosei EA, Touyz RM. immune response to the Thomsen–Friedenreich tumor
2017. Personalized medicine—a modern approach for the antigen (CD176) in mice after vaccination with the com-
diagnosis and management of hypertension. Clin Sci mensal bacterium Bacteroides ovatus D-6. Cancer Immunol
(Lond). 131(22):2671–2685. Immunother. 62(5):875–887.
Schneeberger M, Everard A, Gomez-Valades AG, Matamoros Ulsemer P, Toutounian K, Kressel G, Goletz C, Schmidt J,
S, Ramirez S, Delzenne NM, Gomis R, Claret M, Cani PD. Karsten U, Hahn A, Goletz S. 2016. Impact of oral con-
2015. Akkermansia muciniphila inversely correlates with sumption of heat-treated Bacteroides xylanisolvens DSM
the onset of inflammation, altered adipose tissue metabol- 23964 on the level of natural TFa-specific antibodies in
ism and metabolic disorders during obesity in mice. Sci human adults. Benef Microbes. 7(4):485–500.
Rep. 5:16643. Ulsemer P, Toutounian K, Schmidt J, Karsten U, Goletz S.
Sedwick C. 2014. A novel regulatory T cell population in the 2012. Preliminary safety evaluation of a new Bacteroides
gut. PLoS Biol. 12(4):e1001834. xylanisolvens isolate. Appl Environ Microbiol. 78(2):
Selma MV, Beltran D, Luna MC, Romo-Vaquero M, Garcıa- 528–535.
Villalba R, Mira A, Espın JC, Tomas-Barberan FA. 2017. US Food and Drug Administration. 2016. Early clinical trials
Isolation of human intestinal bacteria capable of produc- with live biotherapeutic products: chemistry, manufactur-
ing the bioactive metabolite isourolithin A from ellagic ing, and control information. https://www.fda.gov/regula-
acid. Front Microbiol. 8:1521. tory-information/search-fda-guidance-documents/early-
Shimbo I, Yamaguchi T, Odaka T, Nakajima K, Koide A, clinical-trials-live-biotherapeutic-products-chemistry-manu-
Koyama H, Saisho H. 2005. Effect of Clostridium butyricum facturing-and-control-information
on fecal flora in Helicobacter pylori eradication therapy. Verma A, Xu K, Du T, Zhu P, Liang Z, Liao S, Zhang J,
World J Gastroenterol. 11(47):7520–7524. Raizada MK, Grant MB, Li Q. 2019. Expression of human
Shinnoh M, Horinaka M, Yasuda T, Yoshikawa S, Morita M, ACE2 in Lactobacillus and beneficial effects in diabetic ret-
Yamada T, Miki T, Sakai T. 2013. Clostridium butyricum inopathy in mice. Mol Ther Methods Clin Dev. 14:161–170.
Wang B, Yao M, Lv L, Ling Z, Li L. 2017. The human micro- Wu TR, Lin CS, Chang CJ, Lin TL, Martel J, Ko YF, Ojcius D, Lu
biota in health and disease. Engineering. 3(1):71–82. CC, Young JD, Lai HC. 2019. Gut commensal
Watterlot L, Rochat T, Sokol H, Cherbuy C, Bouloufa I, Lefevre Parabacteroides goldsteinii plays a predominant role in the
F, Gratadoux J-J, Honvo-Hueto E, Chilmonczyk S, Blugeon anti-obesity effects of polysaccharides isolated from
S, et al. 2010. Intragastric administration of a superoxide Hirsutella sinensis. Gut. 68(2):248–262.
dismutase-producing recombinant Lactobacillus casei BL23 Xu P, Wang Y, Tao L, Wu X, Wu W. 2019. Recombinant
strain attenuates DSS colitis in mice. Int J Food Microbiol. Lactococcus lactis secreting viral protein 1 of enterovirus
144(1):35–41. 71 and its immunogenicity in mice. Biotechnol Lett.
Wells J. 2011. Mucosal vaccination and therapy with genetic- 41(6–7):867–872.
ally modified lactic acid bacteria. Annu Rev Food Sci Yanagibashi T, Hosono A, Oyama A, Tsuda M, Suzuki A,
Technol. 2:423–445. Hachimura S, Takahashi Y, Momose Y, Itoh K, Hirayama K,
Wells JM, Mercenier A. 2008. Mucosal delivery of therapeutic Takahashia K, et al. 2013. IgA production in the large
intestine is modulated by a different mechanism than in
and prophylactic molecules using lactic acid bacteria. Nat
the small intestine: bacteroides acidifaciens promotes IgA
Rev Microbiol. 6(5):349–362.
production in the large intestine by inducing germinal
Woo TDH, Oka K, Takahashi M, Hojo F, Osaki T, Hanawa T,
center formation and increasing the number of IgA þ B
Kurata S, Yonezawa H, Kamiya S. 2011. Inhibition of the
cells. Immunobiology. 218(4):645–651.
cytotoxic effect of Clostridium difficile in vitro by Yang JY, Lee YS, Kim Y, Lee SH, Ryu S, Fukuda S, Hase K,
Clostridium butyricum MIYAIRI 588 strain. J Med Microbiol. Yang CS, Lim HS, Kim MS, et al. 2017. Gut commensal
60(Pt 11):1617–1625. Bacteroides acidifaciens prevents obesity and improves
Wrzosek L, Miquel S, Noordine ML, Bouet S, Chevalier-Curt insulin sensitivity in mice. Mucosal Immunol. 10(1):
MJ, Robert V, Philippe C, Bridonneau C, Cherbuy C, Robbe- 104–116.
Masselot C, et al. 2013. Bacteroides thetaiotaomicron and Zeng L, Tan J, Xue M, Liu L, Wang M, Liang L, Deng J, Chen
Faecalibacterium prausnitzii influence the production of W, Chen Y. 2020. An engineering probiotic producing
mucus glycans and the development of goblet cells in the defensin-5 ameliorating dextran sodium sulfate-induced
colonic epithelium of a gnotobiotic model rodent. BMC mice colitis via Inhibiting NF-kB pathway. J Transl Med.
Biol. 11:61. 18(1):107.

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Critical Reviews in Microbiology

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Next-generation probiotics: a promising approach

Tejinder Pal Singh & Basavaprabhu Haranahalli Natraj

To link to this article: https://doi.org/10.1080/1040841X.2021.1902940

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Next-generation probiotics: a promising approach towards designing

ABSTRACT ARTICLE HISTORY

Introduction probiotics such as designer and next-generation probi-

Figure 1. Changing trends and milestones in the history of probiotics.

Table 1. Signature characteristics of conventional probiotics and next-generation probiotics.

in patients with advanced NSCLC, RCC, or

Viral encephalitis in mice model Ramakrishna

commensals may act as opportunistic pathogens under Selection of potential NGPs

Figure 2. Illustration of the strategies commonly employed for development of NGPs.

producing cells within the upper

Europe, State Food and Drug Administration (SFDA) in

Bohlul et al. (2019)

Overall, the joint expert consultants of FAO/WHO co-

ordinate in assuring the health and nutritional proper-

Cloning and electroporation

(Arora and Baldi 2015).

derived NGPs or engineered probiotics except the gen-

derived from bacterium or yeast need authorization

integrity. Reduced the expression of

transcription factor-kappa B (NF-jB)

inflammation-related molecules and

Recombinant L. lactis producing TRAIL

p65 and its inhibitor IjBa in mice.

Party (BMWP) recommended the Committee for

regulated expression of several

Medicinal Products for Human Use (CHMP) for the regu-

preserved epithelial barrier

latory assessment of biosimilar medicinal products

(EDQM) has framed a Live Biotherapeutic Products

The secreted or cell wall-anchored

Gene coding for human defensin

In the USA, live microbes fall under the category of

pNZ7021 expression vector

foods or dietary supplements when used as foods, or

LBP agents when referring to use as a drug. In this con-

text, based on disease-specific use of commensal

been genetically modified through the purposeful add-

ition, deletion, or modification of genetic material (US

Food and Drug Administration 2016). Furthermore, the

Centre for Biologics Evaluation and Research (CBER) of

the FDA regulates the LBP section. CBER demands strin-

phenotypic and genotypic levels including identity, References

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