Unlocking the Potential of Ghost Probiotics in Combating Antimicrobial Resistance

Abstract

Antimicrobial resistance is a global concern that requires immediate attention. Major causes of development of antimicrobial resistance in microbial cells are overuse of antimicrobials along the food chain especially in livestock, in preventing infections as well as misuse of antimicrobials by patients. Probiotics could be a viable alternative to antibiotics in the fight against antimicrobial resistance. Probiotic strains can act as a complement to antimicrobial therapy, improving antimicrobial function and enhancing immunity. However, there are safety concerns regarding the extensive use of live microbial cells especially in immunocompromised individuals; these include microbial translocation, inhibition of other beneficial microorganisms and development of antimicrobial resistance, among other concerns. Inevitably, ghost probiotics have become the favored alternative as they eliminate the safety and shelf-life problems associated with use of probiotics. Ghost probiotics are non-viable microbial cells (intact or broken) or metabolic products from microorganisms, which when administered in adequate amounts have biologic activity in the host and confer health benefits. Ghost probiotics exert biological effects similar to probiotics. However, the major drawback of using ghost probiotics is that the mechanism of action of these is currently unknown, hence more research is required and regulatory instruments are needed to assure the safety of consumers.

1. Introduction

1.1 Antimicrobial resistance

Misuse and abuse of antimicrobials are key contributors to the introduction of selective pressures in our natural environments, resulting in the rapid increase of antimicrobial resistant microbial strains. Random antimicrobial use has impelled microorganisms to adapt and survive by acquiring antimicrobial resistance genes that lead to antimicrobial resistant strains [1]. Antimicrobials are drugs or medicines, including antibacterials, antivirals, antifungals and antiparasitics, used to prevent and treat infections in humans, plants and animals [2]. Antimicrobial resistance occurs when a microbial strain is no longer susceptible to antimicrobials that would normally inhibit their growth and allows them to withstand the drugs [3]. Chromosomal or plasmid DNA encoding antimicrobial resistance is implicated in the rapid spread of multiple resistance through horizontal gene transfer [4], as shown in Figure 1.

Figure 1.

Mechanisms of horizontal gene transfer in bacteria. Acquired antimicrobial resistance genes can pass between related and unrelated species by transformation, transduction and conjugation.

Mobile genetic elements including plasmids and transposons are instrumental in horizontal gene transfer [5]. Chromosomal resistance is caused by mutations in the developing spontaneous bacterial chromosome [6] while extra chromosomal resistance depends on the extra chromosomal genetic material that can be transferred in ways such as plasmids, transposons and integro [7]. Different types of resistance occur including natural resistance, acquired resistance, cross resistance and multi drug resistance [8, 9].

1.2 Mechanisms of antimicrobial resistance

The major problem with antimicrobial resistance is the selection and stabilization of mechanisms directed by foreign genes taken up by susceptible and resistant strains [2]. Microorganisms can evade the effects of the antimicrobial agents throughdecreased influx (limiting uptake of a drug), alterationof drug target site, drug inactivation using enzymes and active drug efflux (efflux pump) [10, 11] as detailed in Figure 2.

Figure 2.

Mechanisms of antibiotic resistance.

As a result of antimicrobial resistance, antibiotics and other antimicrobial medicines have become ineffective and infections are becoming increasingly difficult or impossible to treat increasing the risk of disease spread, severe illness and death. Alternative strategies are being employed in order to combat antimicrobial resistance. Such strategies include the use of probiotics.

2. Probiotics

Probiotics are live microorganisms, which when administered in correct proportions confer a health benefit on a host [12]. These microbes are a combination of bacteria, fungi, viruses and protozoa [13], and the commonly used probiotics are Lactobacillus and Bifidobacterium [14]. Beneficial probiotics are found in several locations on the body such as the gut, mouth, urinary tract, skin, lungs [8, 15]. Probiotic microorganisms can be isolated from plants, food products, environment, human and animal sources. Probiotics can be administered as supplements in a variety of forms, including in foods, drinks, capsules or pills, powders and liquids [16].

2.1 Characteristics of probiotics

Microorganisms must possess a number of characteristics in order to be classified as probiotics, that is the microbes should be easily isolated from humans, have the ability to live in the gut after consumption, have a proven benefit and must be safe to consume [17, 18]. Some of the characteristics are shown in Figure 3.

Figure 3.

Characteristics of probiotics.

2.2 Mechanisms of action of probiotics

Probiotics exert their biological effect using different mechanisms of action as shown in Figure 4, these include; inhibition of the growth of pathogenic bacteria (competitive exclusion), reduction of bacterial and/or toxin translocation, modulation of the intestinal immune system, production of specific substance such as bacteriocins, modifications of the structure and function of intestinal epithelium, competitive adhesion to epithelial receptors, vitamin absorption and provision of other nutrients [19, 20].

Figure 4.

Mechanisms of action of probiotics.

2.3 Probiotic health effects

Probiotics have a number of health benefits, including lowering the risk of some infectious diseases and reducing the need for antimicrobials to treat secondary infections. For example, the use of probiotics with antimicrobials reduces the incidence, duration and severity of antimicrobial-associated diarrhea, thereby reducing the evolution of resistance [21, 22, 23] and unlike antimicrobials which kill untargeted microbials, probiotics help to keep the gut microbiota in check. Some of the health benefits are shown in Table 1 below.

Probiotic health effects
Metabolic effects	Microbiota & Immunomodulation effects
Reduce risk of colon cancer. Hydrolyse lactose to improve lactose tolerance. Lower levels of mutagenic and/or toxigenic reactions in the gut. Lower serum cholesterol & Supply vitamins like folate to colon epithelium.	Control of irritable bowel syndrome. Colonization resistance which leads to suppression of endogenous & exogenous pathogens e.g., Antibiotic associated diarrhea & travelers’ diarrhea respectively. Strengthened innate immunity& Balanced immune response – to alleviate food allergy symptoms in infants & control inflammatory bowel diseases.

Table 1.

Probiotics beneficial health effects.

Probiotics aren’t perfect, there are many safety challenges associated with the use of probiotics such as ability to acquire antimicrobial resistance and virulence genes [24], chances of microbial translocation from gut to the blood stream [25], high risk of allergic reactions [26] and that biological effects of probiotics are strain specific, therefore proper strain identification is required [27] for a specific condition. As a result, ghost probiotics have become the preferred alternative to probiotics in order to solve the majority of these safety issues [14].

3. Ghost probiotics

Health benefits observed for physiologically active probiotics are not associated with their viability only [28]. Probiotic products containing dead cells can produce effective biological responses. This proves that probiotics merely have an expiry date and can be used beyond that. This phenomenon is known as the probiotic paradox, that is, both live and dead cells produce the same biological response [29]. Ghost probiotics (inactivated probiotics, non-viable probiotics, paraprobiotics) are inactive microbial cells or cell fractions that, when administered in adequate amounts, confer a health benefit to the consumer, [14, 30, 31]. They consist of molecules present on the cell surfaces such as peptidoglycan, teichoic acid, cell wall polysaccharides and cell surface-associated proteins [32]. These trigger the human immune system, stimulating a positive immune response and anti-inflammatory effects in animals and humans [33].

3.1 Technologies used in the production of ghost probiotics

The methods used in producing ghost probiotics are similar to the techniques used for bacterial inactivation such as thermal processing, irradiation, UV rays, high pressure and ultrasound [14, 34] as shown in Table 2 below. Thermal treatment is the most common technique for the production of ghost probiotics in laboratories [14]. The cell membranes are damaged, leading to leakage of nutrients and ions, ribosome aggregation and DNA breakage. Ohmic heating has been proposed for ghost probiotics production. It involves an electric current passing through the sample, leading to fast and uniform heating [35]. Therefore, bacterial inactivation can be caused by thermal and non-thermal damage (electroporation). Inactivation methods have an impact on the beneficial effects. This means that ghost probiotics obtained with different technologies could exhibit different functional features [36].

Methods of cell inactivation	Activities that lead to cell inactivation
Thermal/ heat treatment	Cell membrane damage Leakage of nutrients and ions Protein denaturation Ribosome aggregation DNA breakage
High pressure treatment	Membrane rupture Alteration of ribosomes Protein denaturation & coagulation Reduction of intracellular pH Inactivation of enzymes Loss of solutes
Ultra Violet (UV) Irradiation	Formation of DNA photoproducts
Ionizing radiations	Damage of nucleic acids caused by oxidative radicals
High intensity ultrasound	Cell wall shearing Production of free radicals DNA damage Membrane breakdown & cell lysis

Table 2.

Methods used in the production of ghost probiotics.

3.2 Characteristics of ghost probiotics

They are quite safe, they are well-tolerated and associated with reduced risk for adverse effects in vulnerable individuals [37]. They have no risk for transferring antibiotic-resistant genes to pathogenic or commensal bacteria [38]. Their effectivity is independent of the cell viability, which ensures longer stability and improved shelf-life [39]. They present an easy industrial large-scale production [36]. They provide a wide range of health-promoting effects, some of which can be reinforced in comparison with the effect of intact viable microbial cells [40]. Another very interesting feature of ghost probiotics, is that, due to their nature, it appears feasible that they could be used with concurrent administration with antibiotic and antifungal agents.

Ghost probiotics are categorized into peptidoglycan, teichoic acid, cell wall polysaccharides, cell surface-associated proteins and proteinaceous filaments. These are the ones that mediate beneficial effects to the host [41]. Some bacterial cell walls such as Lactobacilli contain a thick layer of peptidoglycan. This enhances the sensitivity to autolysis, hydrophobicity of the cell envelope and resistance to lysozymes [42]. This part in bacteria can improve innate and systemic adaptive immune responses as well as suppress interleukins which are associated with autoimmune and inflammatory bowel diseases.

Teichoic acids (TAs) are the second main constituent of the cell wall of the microbes. They possess immunomodulatory characteristics and exert anti-inflammatory effects on the intestinal epithelial cells of humans [43]. Cell-wall polysaccharides are common in Gram-positive bacteria surfaces for example exopolysaccharides (EPS). These have the ability to facilitate the interaction of the bacteria with pathogens, have immunoregulatory effects and act as a protective layer [43]. Cell surface proteins are one of the most important components of the outermost cell envelope structure. S-layer proteins, pili proteins, moonlight proteins are part of the surface proteins. These play a role in the host biological processes [44].

3.3 Possible uses of ghost probiotics in combating antimicrobial resistance

To counteract the phenomenon of antimicrobial resistance, there is a need to reduce the frequency in which they are administered. Ghost probiotics are used as a possible solution in fighting against antimicrobial resistance [45]. Due to the risks and concerns of administering probiotics to livestock, scientists are now opting to use ghost probiotics.

Cows tend to suffer from inflammation of the udder (mastitis). The main pathogens that stimulate the infection are Staphylococcus aureus, Streptococcus uberis, isolated strains of Escherichia coli and Streptococcus dysgalactiae [46]. There has been excessive use of intramammary antimicrobials to treat mastitis hence, increasing bacterial resistance. This has reduced their treatment efficacy resulting in a growing interest in replacement therapies without antibiotics such as ghost probiotics [47]. Different variants of ghost probiotics have shown antibacterial activity against S. aureus strains associated with bovine mastitis, increased response of the immune system and a decrease in the number of somatic cells in milk [48].

Increased animal production keeps animals crowded, facilitating the transmission of various diseases. The use of ghost probiotics on farms can naturally bring about a balance of gut microbes, reduce the growth of pathogens and reduce the use of antibiotics for disease prevention [49]. Thus, reducing the occurrence of resistance effects among pathogenic bacteria as the major spread of antimicrobial resistance is through food chains [50]. In a study carried out using Lactobacillus, the cure rate was doubled by the administration of inactivated lactobacilli. For example, the dose of amoxicillin required to kill uropathogenic E. coli was halved [51]. Hence, the use of ghost probiotics can reduce excessive use of antimicrobials. This can help decrease the chances of antibiotic resistance developing.

A variety of ghost probiotics from the Bifidobacterium species have been shown to act as antitumor agents through inhibiting the proliferation of tumor cells. This is still waiting for more human trials for approval. Some have been shown to be cardioprotective, antiulcer, antioxidants and reduce cholesterol in the body [52]. Ghost probiotics containing Bifidobacterium breve and S.thermophilus on preterm infants causes clinical tolerance to tumors necrosis, lowered digestive and respiratory infections, and consumption of these results in lower abdominal distention [53]. Ghost probiotics can treat acute diarrhea, which is one of the most common causes of death in infants and children in developing countries [54]. Ghost probiotic L. paracei can treat atopic dermatitis and other skin infections [55].

There is an innate immune response of macrophages to non-viable Lactobacillus casei cells. This results in an increase in the expression of pro-inflammatory cytokines and an enhanced transcription of toll-like receptors [56]. Ghost probiotics from B. breve and Streptococcus thermophilus cause prolonged dendritic cell survival and maturation hence can be used to enhance immune regulatory function, and improve epithelial barrier function [53]. Lactobacillus paracasei ghost probiotic, can act via the inhibition of immune cell inflammation and protect the host from pathobionts, enteric pathogens and protect a patient against colitis [57].

The capability of ghost probiotics to safeguard the host’s health against serious infections induced by pathogens is fulfilled through various mechanisms such as inhibition of pathogenic adhesion, invasion, biofilm formation, and improvement of immune responses in thegut environment. Additionally, some ghost probiotics derived from Lactobacillus can provide effective protection against infections induced by certain viruses. Lactobacillus species possess antibacterial and anti-viral properties [58], inhibition of Gastric Corona, HIV, and Rotavirus in vitro along with a noticeable diminution in viral load in vivo [6, 30]. According to [59], ghost probiotics have also been shown to fortify endogenous beneficial microorganisms within the gut of the host. This activity by ghost probiotics is suggested to be better than the supplementation with unfamiliar microbial strains as probiotic, [60].

3.4 Advantages of ghost probiotics

To address the safety concerns surrounding probiotics, the attention has switched to the use of non-viable microbial cells, commonly known as ghost probiotics. Ghost probiotic and probiotic cells exhibit similar immunological responses by means of using the same or different mechanisms of action [14, 31, 32]. This has been demonstrated by an experiment done on the human epithelial colorectal adenocarcinoma Caco-2 cell line, both viable and UV-inactivated Lactobacillus rhamnosus GG cells were equally effective in decreasing thepro-inflammatory cytokine, interleukin-8 (IL-8), upon flagellin induction using different mechanisms of action [61]. Additionally, in a different study, RAW 264.7 macrophages were exposed to heat-killed Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus reuteri, Bifidobacterium species, and S. thermophilus as well as the cell envelope components and cytoplasmic extracts of these bacteria. Whole inactivated cells, the cell envelope components, and cytoplasmic fractions from probiotic bacteria stimulated macrophages to producetumor necrosis factor-α (TNF-α), interleukin (IL-6), and nitric oxide [62]. This experiment demonstrated that bioactive compounds responsible for eliciting immune responses can be found anywhere in bacterial cells, and that it does not need an intact cell to elicit immune responses.

Furthermore, various inactivation methods for making ghost probiotics, such as high-pressure treatment and high-intensity ultrasound, have been reported to cause membrane rupture and cell lysis, respectively [14, 63]. Inactivation of microbial cells by cell lysis can produce additional beneficial effects, the contact between the released molecule and the host cells is improved [36, 40], increasing chances of MAMP-PRR interactions which are important for eliciting immunological responses, making ghost probiotics attractive than probiotics.

Misuse of antimicrobials in agriculture and medicine has resulted in development of antimicrobial-resistant microbes in animals. And also, interaction of microbes in gut might result in acquisition of antibiotic resistance and virulence genes in strains that previously lacked these through horizontal gene transfer. Alarmingly, antibiotic-resistant Lactobacillus and Bifidobacterium probiotics have been discovered in dairy products such as milk culture, yogurts, and cheese [27]. As a result of this, the focus has shifted towards using ghost probiotics that cannot acquire and transfer antibiotic resistantand virulence genes. Furthermore, ghost probiotics have a great advantage over probiotics in that they can be administered concurrently with antimicrobial agents without any bioactivity loss and/or development of antimicrobial resistance [64, 65] and thus making it attractive to immunocompromised and multi-diseased individuals.

Industrial processing and storage of probiotic products present viability and stability challenges, probiotic cultures should remain viable and sufficient numbers must reach the target site after thermal processing, storage, and gastrointestinal transit. To avoid these technological challenges, ghost probiotics are used. The dead inactivated cells, ghost probiotics do not require refrigeration to maintain the cultures in a stable and viable state. This reduces the cost of storing and transporting ghost probiotics, allowing them to be used by the poor in impoverished locations such as rural areas where refrigeration machines and facilities are lacking. [14], making them a cheaper and accessible option than probiotics.

Remarkably, ghost probiotics can remain stable in extreme environmental conditions, like water activity (A_w), temperature and pH which are considered stressful to probiotics and they have a longer shelf life. In addition, they can be supplemented into foods, other than dairy products like fruit juices and other cereal products [14], thus provide beneficial effects to lactose intolerant individuals. The heat-inactivated L. gasseri CP2305 strain is an example of ghost probiotics found in non-dairy goods. A sports drink treated with heat-inactivated L. gasseri CP2305 helped young athletes recover from exhaustion, anxiety, and negative moods [66]. Ghost probiotics can withstand the thermal processes during production and thus they can be added before thermal processing without any functionality loss [14]. Because ghost probiotics do not interact directly with food matrices, they have no effect on the organoleptic and/or sensory qualities of the food [34], hence there is no detrimental modification such as that observed in yogurt, which results in high acidification [35].

3.5 Disadvantages of ghost probiotics

Ghost probiotics, being dead and inactive cells, are unable to create metabolites such as bacteriocins, lactic acid, vitamins, and enzymes that are essential for probiotic health effects [14]. Additionally, the chemical mechanism of action of ghost probiotics is unknown; nevertheless, cell wall polysaccharides, peptidoglycans, surface proteins, and teichoic acids are known to activate immunological responses. Unlike postbiotic components which exist in purified form, ghost probiotics mechanism of action is unclear and is difficult to point out which molecule does what due to complex bacterial architecture [36]. Some methods of microbial inactivation such as thermal treatment affect the physiological activity of the resulting dead cells and the stability of their beneficial effects during shelf life, resulting in altered and non-identical biological responses [14, 36]. For instance, heat treatment at 121°C for 15 minutes of multispecies of lactic acid bacteriaie., L. acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, and Enterococcus faecium demonstrated reduced adherent capacity to Caco-2 cells by more than 50% while heat-treatment at 100°C for 30 minutes did not alter the capacity of these strains to adhere to Caco-2 cells at all [67].

4. Current issues surrounding the adoption of ghost probiotics

Their side effects have not been fully understood. Studies have been done on how the microbiome of the gut reconstituted itself after antimicrobial treatment with and without ghost probiotic administration [68]. This means the impact ghost probiotics can have in the medical industry is questionable. There is an issue of, what is being studied is not exactly what would be administered to people [69]. For instance, when research is being carried out it involves a specific organism defined by genus, species and strain (these are pure and carefully dosed). But when buying off the shelf mixed with other products such as food products, people become skeptical about what they are getting dosage wise [70].

Research being carried out is claimed to be of low quality, small in size and often funded by companies with significant conflicts of interest [71].

The inactivation method of ghost probiotics functions can interrupt the bacterial cells and allow for an interface between intracellular bioactive compounds and the host cells on the administration of ghost probiotics. Delivery and formulation of ghost probiotics has been limited in the clinical field [31].

5. Regulation of ghost probiotics

In light of the safety and technological challenges associated with probiotics [14, 40], use of ghost probiotics will expand in near future. Therefore, there is an urgent need to clarify several points to support regulatory authoritiesin defining the requirements for the registration and approval of functional foods containing ghost probiotics and those that have health claims to protect consumers. There is currently an overlap of terminology in defining the biotics terms, ghost probiotics and post biotics which makes communication difficult among researchers, manufacturers, and customers [14]. Therefore, there is a need for internationally recognized clear-cut definitions to avoid confusion that currently exists in biotics, especially for probiotics, postbiotics and ghost probiotics [14]. As result of this mayhem, the ghost probiotics are currently marketed as probiotics [72]. Chiefly, ghost probiotics production, detection, and quantification methods need to be look into closely [14] and standardized [14], before regulations and/or requirements are laid out and implemented. The FDA should then layout the ghost probiotics specific requirements and specifications to iron out the mix-up.

Global commercialization of ghost probiotics is also one of the issues recognized from a regulatory view point because of the geographical differences, for example some traditional probiotics are classified differently across countries like Generally Regarded As Safe for USA and QPS for Europe and additionally some probiotics do not follow the same regulation globally. The regulatory process followed so as to launch a non-traditional probiotic is as complicated as one required for drugs [73].

The current regulations on probiotics are inadequate to protect the consumers and the prescribing doctors, there is abuse of the word probiotics and no specifics of microorganism are indicated in products [74]. Obviously, just like probiotics, ghost probiotics cannot be approved as drugs, even though they are sometimes used for the prevention, management or treatment of disease [75]. In the United States, and many regions of the world, probiotic products are marketed as dietary supplements (not drugs) and are therefore subject to different manufacturing and quality control standards than approved drugs are [75, 76], the same should apply to ghost probiotics. Exemption should be given to ghost probiotics with health claims, these should be treated aspharmaceutical products and regulated as such [75]. To assure safety to end-users, pharmacists should be aware of product quality when recommending these dietary supplements to risk populations like immunocompromised individuals [75] and infants and manufacturing quality control standards should be steeper especial for this vulnerable group [40].

Additionally, manufacturers should be in a position to provide evidence of quality criteriawhen required to and they should guide pharmacists on the safe use of specific products [75]. Manufacturers should have quality management systems in place, and third-party and/or regulatory organizations should verify compliance. Accordingly, the regulatory aspects that need to be considered for ghost probiotics are efficacy, safety, andquality control of manufacturing.

6. Future perspectives and conclusion

There is a need for large randomized placebo-controlled single strain trials with standard dosing, formulation and duration of treatment in various diseases to get consistent results. At this moment it is difficult to recommend any particular ghost probiotic for a particular disease as the preparation and dosing may not be available commercially. The interaction of the gut microbiota with its host and mutual regulation has become one of the important topics of biomedical research. Their relevance in human diseases require much more research. The popularity of ghost probiotics is fast increasing shortly they will be used in food, medicine, and agriculture. Additionally, the diet microbiota host interface can give rise to newer therapeutic approaches based on selective alteration of microbial metabolite production to support human health and prevent diseases. The metabolic profiling approach, suggests how mining the microbiota may lead to personalized treatment.

Conflict of interest

The authors declare no conflict of interest.