Probiotics " Alternative to antibiotics resistance"

Probiotics " Alternative to antibiotics resistance"

Walaa, I. Ahmed and AL-Shimaa , A . Sleim

 Agriculture Research Center,Animal Health Research Institute.

 Alexandria, Bacteriology department

Antibiotic resistance is of  great concern  for human and animal globally. New resistance mechanisms are emerging and can spread between food-producing animals and humans all parts of the world . Antibiotic resistance is threatening our ability to treat common infectious diseases. Antibiotic resistance is accelerated by the misuse and overuse of antibiotics , so there is great demand for researches  to discover alternative substances to reduce the impact and limit the spread of resistance. Probiotics are considered to be favorable live microorganisms by the host organism by maintaining microbial homeostasis and healthy gut, morover, probiotics have been suggested as viable alternates to antibiotics and subsequently overcome antibiotic resistance and reduce the excessive use of antibiotics. In addition, providing other growth-promoting properties in the animal health and nutrition industry.

Introduction:                        

Probiotic in Greek language means for life’ The first probiotic definition was reported in 1908 by Élie Metchnikoff  who noticed that consumption of fermented dairy products enhance  the health life. He then reported that the presence of lactic acid bacteria in fermented dairy products helps keep the defensive system activated, resulting in higher longevity of its consumers (Stambler,2017).WHO and FAO in 2002 , mentioned that probiotics are live microorganisms that revealed health benefits to the host when ingested in adequate amounts. According to the latest definition of the WHO, probiotics are refer to active microbes that stimulate the growth of other probiotic bacteria in the gut and possess beneficial health effects to the host (Hill et al.,2014). Guarner and Malagelada, (2003) pointed out that Lactic acid bacteria and bifidobacteria are the most strains frequently used as probiotics. Several microorganisms, particularly bacteria and fungi, have habored probiotic activities , species belonging to the genera  Lactobacillus,  Streptococcus,  Lactococcus, and Bifidobacterium remain the most popular probiotic agents to date (Hoseinifar et al.,2018).

There are records showing that the use of bacterial probiotics are more effective in chickens, pigs, and young calves, however, probiotic yeast (Saccharomyces cerevisiae) and fungal strains (Aspergillus oryzae) reveals better results in adult ruminants (Markowiak and Śliżewska .,2018).

Probiotic microorganisms:

There are many different microorganisms used as probiotics . Bacteria, bacteriophages, microalgae, and yeasts are all examples of probiotics (Llewellyn etal.,2014). There are numerous  specific types of bacteria  which consider as common probiotics commonly used in the livestock to date, which are Lactobacillus , Bifidobacterium, Streptococcus,  Enterococcus,  and Lactococcus (Collado etal.,2005 ; Llewellyn etal.,2014; and Abdelqader etal.,2013). Probiotics are also made up of good yeast. The most common types of yeast found in probiotics is Saccharomyces(S. cerevisiae and S. bourlardii), Candida pintolopesii, and Aspergillus oryzae are typical non-bacterial probiotics (Abdelqader etal.,2013, Mookiah etal.,2014 and , Pedroso etal.2013).

-  Lactobacillus species :

Lactobacillus species is a type of bacteria used therapeutically as probiotics . There are lots of different species of lactobacillus. Lactobacillus species  are   Gram-positive rods  , obligate and facultative anaerobes in the human gastrointestinal and genitourinary tracts (Fujisawa et al.,1992 McGroarty ,1993 and Madsen et al.,1999). lactic acid–producing as the major metabolic end product of carbohydrate fermentation. They also help in the breakdown of foods, therefore producing hydrogen peroxide, lactic acid, and other substances which create an acidic, unfavorable environment for harmful or pathogenic organisms. Lactobacillus bacteria are commonly found in the human gut, mouth and vagina. They are considered generally as “good bacteria”, and in fact may contribute to good health. Lactobacillus is important for gastrointestinal function, as they are involved in vitamin B complex and vitamin K synthesis, natural antibiotic production, enhance innate and acquired immune defense, digestion and metabolize proteins and carbohydrates, detoxification of pro-carcinogens.

Some researchers have demonstrated that Lactobacillus can produce antifungal substances, such as benzoic acid, methylhydantoin, mevalonolactone (Prema  et al .,2008 and Niku-Paavola etal.,1999) and short-chain fatty acids (Sjogren et al .,2003). Magnusson and Schnürer (2001) discovered that  Lactobacillus coryniformis can produce proteinaceous compounds exhibiting antifungal properties. Lactobacillus reuteri is one of the well-documented probiotic species in lactic acid bacteria and is mainly found in Gram-positive bacterium in the gut flora of animals and birds (Bhogoju etal.,2018). Lactic acid bacteria  are generally recognized as safe due to their ubiquitous appearance in food and their contribution to the healthy microbiota of animal and human mucosal surfaces. Due to their beneficial and nonpathogenic effects, Lactic acid bacteria  are considered to be potential probiotics.

- Bifidobacterium species:

Bifidobacterium is an anaerobic, Gram-positive, V- or Y-type branched, rod-shaped, immobile, non-spore-forming, Gram-positive, anaerobic, catalase-negative bacteria that belong to the family Bifidobacteriaceae and the phylum Actinobacteria. Bacteria in the Bifidobacterium genus can utilize glucose and produce lactic and acetic acids as by-products . Bifidobacterium are found in large numbers in the gut of animals and human. Many Bifidobacterium are generally used as probiotics in human food and in pharmaceutical formulations (Gaggìa etal.,2010). Bifidobacterium pseudolongum have shown significant results on a better food conversion ratio (FCR)in piglets with no differences in final weight, weight gain, and feed intake , Moreover  many of these germs have “GRAS” (“Generally Regarded As Safe”) status (Afonso etal.,2013).

- Bacillus species:

Bacillus is a genus of Gram-positive, aerobic or facultative anaerobic, endospore-forming bacteria. The ability to form spores is useful  and provides  long-term storage without the loss of viability compared to those containing non spore-forming bacterium. Inaddition, spores are able to survive the harsh, low pH of the gastric barrier and can reach the small intestine to perform their probiotic properties (Cutting 2011). Now many strains of some Bacillus species are used as probiotic dietary supplements in animal feeds. Bacteria of the genus Bacillus already used as probiotics have real potential and can be used in safe production and as an alternative to conventional antibiotics. Several researchers showed that these species possess high potential for immunomodulation and protection against diseases in animal breeding, and recommend Bacillus subtilis as a beneficial agent for the biological control of the diseases (Hoseinifar  etal.,2018). Animal model research mentioned that ingesting bacillus spores increases immune response (Duc et al .,2004).

- Saccharomyces species:

Saccharomyces is a genus of budding yeast; it is also part of the gut microbiota. S. cerevisiae var. boulardii is an eukaryotic organism that has been used in scientific investigations since the time of its discovery (Khaneghah etal.,2020). Saccharomyces cerevisiae var.  boulardii  is the most significant probiotic yeast species  due to its ability to produce different bioactive compounds (Lazo-Vélez etal.,2018). It is a non pathogenic yeast strain that has been used for the treatment and prevention of diarrhea. Saccharomyces cerevisiae var. boulardii  is best known for its role in treating gastrointestinal diseases ( Batista et al.,2014 and  Drozdova et al.,2016). Other species belonging to this genus such as Saccharomyces carlsbergensis are also used as probiotics in animal feeding (Gaggìa etal.,2010).

- Lactococcus species:

Hoseinifar  etal.,(2018) reported that Lactococcus lactis  was capable of protecting different fish species against bacterial pathogens . Lactococcus strains are commonly used in the manufacture of fermented dairy products. Although  Lactococcus lactis is considered safe for human and animal use, some studies have also linked  Lactococcus bacteria (Lactococcus lactis and Lactococcus garvieae) to infection (Rodrigues etal.,2016).

- Enterococcus species:

Enterococcus is a common member of the endogenous intestinal microbiota of humans and animals (Araújo and Célia,2013). Sometimes, these strains are involved in the production of substances such as β-hemolysin, gelatinase, and aggregation substance that have undesirable phenotypes in probiotic strains (Araújo and Célia,2013). Although this genus is not considered “generally recognized as safe”, species from the genus Enterococcus have been used as probiotic for human or animals (Araújo and Célia,2013)and (Sivieri etal.,2008). These bacteria may participate in transmission of resistance to antibiotics (Anadón etal.,2006).

- Streptomyces species:

 Streptomyces is mainly used as a probiotic in aquaculture, because of its unique ability to produce several antimicrobial agents as secondary metabolites. Several promising results of the genus Streptomyces was reported as probiotics in aquaculture ( Das et al.,(2010) and Augustine et al.,(2015)).

- Microalgae:

Recently , microalgae have more attention due to their wide range of nutritionally important compounds for humans and animals, including polysaccharides, polyunsaturated fatty acids (PUFA), proteins, and antioxidants, such as carotenoids and phenolic compounds (Sathasivam etal.,2019). The actual and prospective clinical applications of microalgae raise the possibility that they can be used as probiotics , due to their ability to normalize the functioning of the microbiota of humans and agricultural animals and its ability to produce biologically active substances, including hormones, neurotransmitters, and immunostimulators. Several research investigated  the  probiotic properties of microalgae have shown that  aqueous  algae  extract from  Spirulina platensis, Chlorella, Dunaliella salina, Chlorococcum are potential sources. In food and nutrition biotechnology, algae are usually used as extracts that can improve probiotic performance. Recently, cultivation of live microalgae with probiotics has gained more attention because interspecies interactions can add more value to the final product.

* Spirulina :

Spirulina belongs to blue-green autotrophic microalgal, which contains about 70 % of protein, being also rich in unsaturated fatty acids and pigments, such as linolenic acid and linoleic acid, carotenoids and chlorophylls (Bezerra et al., 2020; Li et al., 2019). Spirulina is commonly present in  many  freshwater  environments. Spirulina is produced on a large scale in outdoor pools for commercial purposes to be used as a nutritional  supplement  in  some  countries  such  as  Thailand,China,  the  United  States,  and  India. On the other hand, * * * * Chlorella:

Chlorella is a single-celled green microalgae that can exist in fresh and sea water (Eckardt, 2010).Chlorella belongs to the “Generally Recognized As Safe” (GRAS) category of the US Food and Drug Administration being rated as a green and healthy food by the Food and Agriculture Organization of the United Nations (FAO) (Song et al., 2018). Recent studies have reported that the biologically active ingredients in Chlorella show positive effects as anti-hypertensive, anti-allergic, anti-asthmatic, anti-diabetic, anti-tumor and preventing heart disease (Barboríkov´ a et al., 2019; Horii et al., 2019).

From the perspective of anti-inflammatory and microbial growth curves, the aqueous extracts of Spirulina, Chlorella and P. tricornutum may be suggested as potential sources of natural anti inflammatory agents and antimicrobials for the prevention, treatment and control of bacterial infections and stimulation of probiotic activity (Jianjun  etal.,2023).

* Dunaliella:

Dunaliella, together with other microalgae, Arthrospira  and  Chlorella, have been approved by the Food and Drug Administration (FDA) as a food source with a Generally Recognized as Safe (GRAS) status (Sui etal.,2020). Although Dunaliella salina is not as popular as Chlorella and Arthrospira, it has many beneficial properties for the development of new functional foods with probiotics and novel therapeutics(Ivana etal.,2022).

* Chlorococcum:

Chlorococcum sp. are non-toxic microalgae, and they are found in poorly studied and isolated regions (Tanmay etal.,2021).  Chlorococcum sp. can accumulate nutrients, especially carbohydrates, lipids, and bioactive compounds of interest (Clediana etal.,2016). 

- Bacteriophages:

First discover of bacteriophages was by the British bacteriologist William Twort  in 1915 . In 1917 the French-Canadian microbiologist Felix d’Herelle, realized the presence of some biological entities having the ability to kill bacteria. D’Herelle named them “bacteriophages” to refer that these viruses were able to “eat” and “devour” bacteria (Sulakvelidze etal.,2001). Phages have been played a role in controlling infectious diseases in aquaculture, moreover  control diseases associated with severe economic losses (Sieiro etal.,2020 and Nakai etal.,2002). Several studies  have been showed the effective control of fish diseases caused by several species belonging to the genus Aeromonas species. (Akmal etal., and Kim et al., 2015) or Vibrio species (Matamp etal., 2020;Khatharios etal.,2017;and Chen etal.,2019) , highlighting the effectiveness of phage treatment as an excellent alternative to antibiotic treatment.  Phages have been mentioned as a feasible alternative for treatment and prophylaxis in cattle, where the most prevalent infectious diseases either clinical or subclinical mastitis, metritis or respiratory infections caused by bacterial agents (Gutierrez etal.,2019).

rella, Dunaliella salina, Chlorococcum are potential sources.

  Pros effects of probiotics:

Probiotics have demonstrated several important effects as therapeutic options for a variety of diseases in addition to enhance nutritional value of food products, improve the immune system, prevent gut infections , suppress antibiotic-associated diarrhea, and  reduce lactose intolerance symptoms, moreover, reduction of colon cancer risk.One of the most benefical proposed mechanism of probiotics is the formation of antibacterial substance termed bacteriocins and production of organic acids such as lactic and acetic acids which consider as the main antimicrobial compounds responsible for the inhibitory activity of probiotics against pathogens.The action of organic acids of probiotics against pathogens occured by entering of undissociated form of organic acids into the bacterial cell and then dissociates inside bacterial cytoplasm leading to lowering of the intracellular pH or the accumulation the ionized form of organic acid intracellular of the pathogen can lead to  the death of the pathogen (Ouwehand,1998 and Russelland Gonzalez .,1998). Bacteriocin may enable the direct inhibition of pathogen growth within the gastrointestinal tract (O’Shea.,2014). Also, probiotic bacteria are able to produce de-conjugated bile acids, which are derivatives of bile salts. De-conjugated bile acids exhibit  a stronger antimicrobial activity compared to that of the synthesized bile salts of  the host organism, So probiotics protect themselves from their own bactericidal metabolites (Oelschlaeger ,2010).Moreover, some strains of probiotics have the ability to produce metabolites that inhibit fungi growth(Coloretti etal.,2007 , Lindgren and Dobrogosz1990).

Benefit of probiotics in animal:

There is an evidence that the use of probiotics instead of antibiotics is benefical  in  promoting animal growth through increase digestive enzyme, increase feed intake, increase body weight gain and increase reproductive performance( Zhang et al.,2021 and Lokapirnasari etal.,2019).Some  probiotic strains have the ability to inhibit bad effect of  animal pathogens as they have the ability to reduce infection and disease , increase resistance to pathogen , enhance immune response, improve intestinal microbiota and enhance antioxidant status . and may be considered as antibiotic alternatives in poultry, swine, cattle farming and others for enhancing immune function and disease prevention (Vieco-Saiz etal.,2019). Administration of probiotics has shown increased effect in the levels of immunoglobulins such as M and A along with the increased levels of total antioxidant capacity in serum (Wang et al.,2018)  Few probiotic strains have anti-inflammatory properties which allow the balance between pro- and anti-inflammatory cytokines  (Cristofori et al.,2021 and , Pagnini et al.,2010), Along with the anti-inflammatory responses, probiotics also exhibit anti-viral properties in animals (Lehtoranta etal.,2020).

 Probiotics have ability for the production of antimicrobial substances, such as bacteriocins, hydrogen peroxide, and volatile fatty acids (Vieco-Saiz etal.,2019). It was recorded that organic compounds produced by probiotic bacteria have proven to exhibit inhibitory effects against pathogenic bacteria such as Helobacter pylori (Rezaee etal.,2019). Ma et al.,(2019) reported that probiotic microbes such as Bacillus subtilis, Saccharamyces cerevisiae, and Enterococcus faecalis can enrich milk secretion in cows . Mammary gland condition improved along with the improved functions of the teat sphincter were noticed by the effect of Lactobacillus base teat spray (Alawneh etal.,2020).Addition of Rhodopseudomonas palustris showed improved microbial fermentation and high rumen microbial growth performance (Chen et al. ,2020).  

Charcters of  antibiotic and probiotic:

Safety of Probiotics:

A bacterial strain’s safety, its source, antibiotic-resistant characteristics, and absolute lack of pathogenicity associated with virulent cultures all contribute to the safety profile’s primary foundation; the rest is performance (Galdeano and Perdiagon ,2004). The majority of probiotics are safe. Nevertheless, adverse effects have been sporadically reported and caution of potential side effects should be taken. A demonstrative report about probiotics and their potential side effect has been published by WHO/FAO(2002). Probiotic strains should be characterized by the absence of their virulent profile and their low resistance to antibiotics. Probiotics have a long record of safety use during history, which relates primarily to the use of Lactobacilli, Bifidobacteria, and Streptococcus (Shanahan ,2012) . New probiotic microbes should belong to genera and strains commonly found in the healthy human intestinal microflora or in dairy food products. Accurate bacterial identification represents a critical step in evaluation of the safety of  new probiotic strains (Holzapfel et al.,2001). Andrighetto et al.,1998 recorded that microbial identification at the molecular level should be applied because physiological and/or biochemical characterization alone is insufficient to achieve a reliable identification.

Probiotic performance promotes a variety of pathways, including adhesion to epithelial cells, decrease in gastrointestinal permeability, and immunoregulatory impacts (Lee and Salminen ,2008). Probiotics are not metabolized, have no potential for transference to animal-derived foods, and so do not result in the creation of residues. Due to the absence of their explicit and/or indirect transit from the gut into the animal body, they do not affect metabolic activities and therefore have no adverse effect (Fefana,2021). The introduction of novel microbes needs an acute investigation and assessment of their safety and the risk-to-benefit ratio.

Conclusion:

The excessive and uncontrolled use of antibiotics are associated with the emergence of antimicrobial resistance. Continuous rising in multidrug-resistant organisms is responsible for causing millions of deaths and economic loss all over the world annually. Spreading of antimicrobial resistance led scientists to search for alternative solutions to antibiotics to overcome the global problem of several pathogens’ resistance for both human and animal infections. Although probiotics is one of these solutions that can inhibit pathogens by production of toxic compounds, modulation of the immune system  or competition for a colonization sites or nutritional sources , in additon, several scientific  studies report the potential use of several probiotics as an alternative to antibiotics and their results seem very promising , despite some technical issues that should be solved before implentation , so probiotics need more research to gain additional data on their mode of action to improve its efficacy and verified how they whether benefit or not to the host with a practical approach without impacting on environmental pollution or antimicrobial resistance ..