Microorganisms with Claimed Probiotic Properties: An Overview of Recent Literature

Probiotics are defined as live microorganisms, which when administered in adequate amounts, confer a health benefit on the host. Health benefits have mainly been demonstrated for specific probiotic strains of the following genera: Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, Escherichia coli. The human microbiota is getting a lot of attention today and research has already demonstrated that alteration of this microbiota may have far-reaching consequences. One of the possible routes for correcting dysbiosis is by consuming probiotics. The credibility of specific health claims of probiotics and their safety must be established through science-based clinical studies. This overview summarizes the most commonly used probiotic microorganisms and their demonstrated health claims. As probiotic properties have been shown to be strain specific, accurate identification of particular strains is also very important. On the other hand, it is also demonstrated that the use of various probiotics for immunocompromised patients or patients with a leaky gut has also yielded infections, sepsis, fungemia, bacteraemia. Although the vast majority of probiotics that are used today are generally regarded as safe and beneficial for healthy individuals, caution in selecting and monitoring of probiotics for patients is needed and complete consideration of risk-benefit ratio before prescribing is recommended.

also adopted by the International Scientific Association for Probiotics and Prebiotics (ISAPP) and is used in most scientific publications. However, this definition is not accepted by European Food Safety Authority (EFSA) or the U.S. Food and Drug Administration (FDA) [13,14] at the moment since they insist that the health claim incorporated in the definition is not measurable due to the fact that commercial markets have outpaced the ability of science to substantiate the evidence. As a consequence, no substantiated health claims have been approved for any probiotic in the U.S. [15]. However, health claims, such as treatment and cures of disease using probiotics, are measurable and can be proven with similar type of studies as are conducted for drugs (double-blinded, randomized, placebo-controlled human trials). Although the benefits of consuming fermented foods have been known to humankind for centuries; long before microorganisms were discovered; the concept of administering microorganisms in order to confer a positive health benefit started over a century ago when Metchnikoff theorized that health could be enhanced, and also senility could be delayed, by manipulating the intestinal microbiome with host-friendly bacteria found in yogurt [16].
Over the last two decades there has been growing interest on both basic and clinical science in probiotics which has resulted in over 6000 publications in the biomedical literature, with over 60% published in the last 5 years, some in the top ranking scientific journals [14]. The most common types of microorganisms used as probiotics are lactic acid bacteria and bifidobacteria, although other bacteria and certain yeasts are also used [17]. It is important to stress that the biological effects of probiotics are strain specific and that the success or failure of one strain cannot be extrapolated to another strain. Thus proper strain identification using novel molecular and based technologies is imperative [18]. Species identification can be performed by 16S rRNA gene sequence analysis and DNA-DNA hybridisation techniques. Strain identification can further be performed by various reproducible molecular methods or using unique phenotypic traits. Examples of molecular methods are: pulsed field gel electrophoresis and randomly amplified polymorphic DNA. Phenotype traits for strain identification include: determining the presence of extrachromosomal genetic elements, fermentation of a range of sugars and detection of final fermentation products obtained from glucose utilization [12].
Another important aspect to mention when claiming the health benefits of probiotics is that a beneficial effect can only be demonstrated by in vivo studies [12]. Although in vitro studies or animal models cannot prove a probiotic effect, they can be used to characterise a possible mechanism of probiotic action, determine the safety of probiotic microorganisms or convey other knowledge of probiotic strains. Thus, in vitro studies provide the first step in evaluating probiotics for food use and should be followed by double-blinded, randomized, placebo-controlled human trials. Appropriate target-specific in vitro studies that correlate with in vivo are recommended. For example in vitro bile salts resistance was shown to correlate with gastric survival in vivo [19]. Knowledge on probiotic strains is therefore the first step conducted through in vitro studies. The most important properties of strains to even be considered for probiotic use include: resistance to gastric acidity, bile acid resistance, adherence to mucus and/or human epithelial cells and cell lines, antimicrobial activity against potentially pathogenic bacteria or fungi, ability to reduce pathogen adhesion to surfaces, bile salt hydrolase activity, enhancing viability of probiotics. [12,[20][21][22][23][24].
The aim of this overview of recent literature was to present the most common microorganisms with their recently claimed probiotic properties.

Methods
A literature overview of the PubMed database using the keywords: 'probiotic' and 'microorganisms' yielded 920 publications. Of these; 682 were published in the last 10 years (74 %); and 492 were published in the last 5 years (53 %). Thomson Reuters Web of science yielded 1078 publications using the keywords: 'probiotic' and 'microorganisms' since 1970, of these 886 (82.1 %) were published in the last 10 years and 676 (62.7 %) were published in the last five years. The literature overview for this article included other databases such as ScienceDirect and Google search as well as other keyword combinations (individual microorganism/probiotic). Unless otherwise relevant, only a selection of the most recent articles focused on probiotic microorganisms and their health claims were chosen to be included in this overview. Of course, this does not mean that all important research on probiotic microorganisms was included.

Specific Strains with Probiotic Properties in the Genus Lactobacillus
The genus Lactobacillus includes various Gram positive facultative anaerobic or microaerophilic rod-shaped bacteria. They are a major part of the lactic acid bacteria (LAB) group (including Lactobacillus, Lactococcus, Enterococcus, Oenococcus, Pediococcus, Streptococcus and Leuconostoc species) that can convert hexose sugars to lactic acid thus producing an acid environment which inhibits the growth of several species of harmful bacteria [25]. In humans, Lactobacilli are normally present in the vagina, gastrointestinal tract [26] and are together with Bifidobacterium one of the first bacteria to colonize the infant gut after delivery [27]. The complete genome sequences of the following commensal Lactobacilli have been published: Lactobacillus plantarum, L. johnsonii, L. acidophilus, L. sakei, L. bulgaricus, L. salivarius [25].
Some Lactobacilli are used for the production of yogurt, cheese, sauerkraut, pickles, sourdough, wine and other fermented products [28][29][30]. In all cases, sugars are metabolised into lactic acid; thus creating a hostile environment for spoilage microorganisms and enabling food preservation.
It has been found that infants with food allergies exhibit an imbalance between beneficial and potentially harmful bacteria, i.e., decreased Lactobacillus, Bifidobacteria and Enterococcus species and increased coliforms, Staphylococcus aureus and Clostridium species; suggesting that microbial inhabitants of the human body, may play either a pathogenic or protective role in allergies. Based on this data, many clinical trials addressing the use of probiotics in the context of allergic disorders have been conducted in children. However, currently, no conclusive item may be drawn [31].
Lactobacilli have received tremendous attention due to their health-promoting properties [26]. However, a very important fact that is sometimes overlooked by scientists is that most Lactobacilli do not form stable and numerically significant populations in the human intestinal tract, especially in the small intestine where they are presumed to form epithelial associations [26]. Similarly, in the research on modulating intestinal bifidobacteria and Lactobacilli after one week supplementation with commercial probiotic food supplements, only a significant increase in the intestinal Lactobacillus acidophilus group was observed and even this increase disappeared after a few days [32]. The authors concluded that short-term daily intake of live probiotic cells is insufficient in modulating the intestinal bifidobacteria and Lactobacilli. Lactobacilli such as: Lactobacillus acidophilus, L. casei, L. paracasei, L. rhamnosus, L. delbrueckii subsp. bulgaricus, L. brevis, L. johnsonii, L. plantarum and L. fermentum are commonly used as probiotic products. Although Lactobacilli are often described as indigenous inhabitants of the human intestinal tract, they are more likely to be autochthonous of the oral cavity or fermented foods [26,32].
Studies have shown that certain strains of Lactobacilli are effective in preventing antibiotic-associated diarrhoea [33,34]. Lactobacilli species are commonly selected as probiotics since they express many crucial properties such as: high tolerance to acid and bile, capability to adhere to intestinal surfaces, withstanding low pH, gastric juice, inhibiting potentially pathogenic species (antimicrobial activity), resisting antibiotics, producing exopolysaccharides and removing cholesterol [20,35,36]. Lactobacillus rhamnosus CRL1505 has even been effective in reducing viral-associated pulmonary damage through controlling immune-coagulative responses and clearing respiratory viruses [37]. In a published meta-analysis [38] it has been shown that even though the probiotic strains of Lactobacillus were safe and effective in preventing recurrent urinary tract infections in adult women, the authors concluded that more randomized clinical trials should be conducted to make a more definitive recommendation.
Sometimes probiotic 'cocktails' comprising of various strains are used [39][40][41]. Several probiotic strains of microorganisms are effective in competing against common causes of travellers' diarrhoea [39,42] caused mostly by bacterial pathogens such as: one of the seven types of diarrheagenic Escherichia coli, Campylobacter jejuni and Shigella species. The most commonly used probiotic microorganisms against these pathogens are: Lactobacillus acidophilus, Lactobacillus rhamnosus GG, Saccharomyces boulardii, Bifidobacterium bifidum and Bacillus coagulans. Using Lactobacilli and other probiotics together with antibiotics also seems to be promising for the treatment of bacterial vaginosis, although more well-designed randomized controlled trials with standard methodologies and larger patient size are needed to inconclusively prove benefits [39].
The review of randomized control trials on the use of probiotics for functional constipation [43] revealed that the favourable treatment for adults was with Bifidobacterium lactis DN-173 010, Lactobacillus casei Shirota and Escherichia coli Nissle 1917. A beneficial effect on children was shown with Lactobacillus casei rhamnosus Lcr35. In the review of probiotic safety during pregnancy [41] it was found that Lactobacillus and Bifidobacterium had no effect on the incidence of Caesarean section, birth weight or gestational age and no malformations were reported. The study [44] monitoring one month administration of a multi-strain probiotic capsule containing: Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium animalis subsp. lactis and Streptococcus thermophilus gave indecisive results, as no difference in the colonic microflora of patients with colitis (Crohn's colitis or ulcerative colitis) was observed.
On the other hand, a liver abscess caused by a Lactobacillus rhamnosus that was not distinguishable from the probiotic strain L. rhamnosus GG [45] and Lactobacillus endocarditis caused by L. rhamnosus (strain not specified) have been reported [46]. Although both reports [45,46] gave no information on the origin of infection; both reports highlight the importance of strain identification. It is also important to add that both articles were dated from the year 1999 when methods for exact strain identification were not as perfected as today.

Specific Strains with Probiotic Properties in the Genus Bifidobacterium
The genus Bifidobacterium includes various Gram positive non-motile anaerobic bacteria. They are endosymbiotic inhabitants of the gastrointestinal tract and vagina of mammals, including humans [47]. Strains of the genus Bifidobacterium are also often used as probiotic bacteria as they are known for their variety of resistance mechanisms to bile salts, which is important since the beneficial effects of probiotic bacteria must be generated in the presence of this biological fluid.
It has even been proven that although bile tolerance is strain dependent, both wild type-bile sensitive bifidobacteria and Lactobacilli strains can progressively adapt to the presence of bile salts by subculturing and gradually increasing concentration of bile [36,48]. Several strains of bifidobacteria are considered as important probiotics including: Bifidobacterium infantis, B. adolescentis, B. animalis subsp animalis, B. animalis subsp lactis, B. bifidum, B. longum, B. breve. Companies often use trademark names for some of these bifidobacteria by inventing scientific sounding commercial names.
As mentioned before Bifidobacterium species together with other probiotics have been proven to treat constipation [43], travellers' diarrhoea [39], antibiotic-associated diarrhoea [34], maintaining remission of disease activity of gut inflammation and moderate ulcerative colitis [49,50], prevention as well as treatment of necrotizing enterocolitis in newborns [51], reduction of radiation induced diarrhoea [52], reducing the development of disease risk for eczema, food allergies [53], cholesterol-lowering capacities [36].

Specific Strains with Probiotic Properties in the Genus Saccharomyces
The genus Saccharomyces includes various yeasts such as: Saccharomyces cerevisiae (used for making wine, bread, beer), Saccharomyces bayanus (used for making wine) and Saccharomyces boulardii used in medicine as a probiotic. Saccharomyces yeasts also form symbiotic matrices with bacteria to form kefir [54] and are sometimes a component of kombucha [55].
S. boulardii is often marketed as a probiotic in a lyophilized form to treat diarrhoea while maintaining an excellent reputation for safety [39]. Most reports show a clinical benefit of S. boulardii in decreasing the duration of diarrhoea regardless of the cause and thus reducing hospital stay resulting in social and economic benefits [7,33,[56][57][58]. Administration of S. boulardii has shown positive effects for patients with irritable bowel syndrome [59], preventing and treating relapses of inflammatory bowel disease and for treating moderate symptoms of ulcerative colitis [60,61]. Recurrent pseudomembranous colitis infection caused by Clostridium difficile can also be significantly reduced by administration of daily dosages of S. boulardii together with standard antibiotics [62]. In the previously mentioned review of probiotic safety during pregnancy [41] no malformations were reported. On the other hand, it must not be neglected that in immunocompromised individuals or other patients, S. boulardii can cause fungemia or localised infections [63,64].

Specific Strains with Probiotic Properties in the Genus Lactococcus
Lactococcus is a genus of Gram positive lactic acid bacteria that are commonly used in the dairy industry for manufacturing fermented products. They are important in preventing growth of spoilage bacteria in milk products due to acidification. They are also sometimes recommended as probiotics.
Certain strains of Lactococcus lactis subsp. lactis have probiotic properties such as adhesion to vaginal epithelial cells and nisin production (Lactococcus lactis subsp. lactis CV56) [65,66] and are also used to treat antibiotic-associated diarrhoea in combination with other probiotics [33].

Specific Strains with Probiotic Properties in the Genera Streptococcus and Enterococcus
The genera Streptococcus and Enterococcus are also part of the lactic acid bacteria and contain several strains associated with severe health-care associated infections such as: Streptococcus pyogenes, Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecium [67]. However, other strains form part of the commensal human microbiome of the mouth, skin, and intestine, such as Enterococcus faecium PC4.1 [68] and others. Some strains have probiotic properties: such as Enterococcus durans [69] and Streptococcus thermophilus [70] (also used for the production of yogurt alongside Lactobacillus delbrueckii subsp. bulgaricus).
Although Enterococcus faecium has a long history of probiotic use, especially in preventing antibiotic-associated diarrhoea [34], certain strains are opportunistic pathogens that present a potential reservoir of antibiotic resistance and virulence genes (animal study) [71] and are therefore generally not treated as safe (GRAS) for humans, but represent important probiotics for animals [72,73].

Specific Strains with Probiotic Properties in the Genus Bacillus
The genus Bacillus includes Gram positive spore-forming aerobic or facultative aerobic members with claimed probiotic properties including: B. subtilis, B. coagulans, B. subtilis, B. cereus.
Bacillus coagulans together with other microorganisms has proven to be most successful in preventing or treating antibiotic-associated diarrhoea [34,42].
Bacillus subtilis spores have been considered as probiotics for animal consumption [74,75] and have been proposed for treating diarrhoea and H. pylori eradication in humans [76]. However, a report of a recurrent septicaemia in an immunocompromised patient due to treatment with spores of the probiotic strain B. subtilis [77] and four cases of nosocomial bacteraemia caused by absorption of an oral preparation containing B. subtilis spores [78] have shown the high risk of using Bacillus subtilis spores as probiotics to immunocompromised patients, and that ingestion is safe only for humans under normal host conditions [79].
B. cereus NVH 75/95 has also proven to be an efficient probiotic for animals [80]. Certain B. cereus strains are human-lethal and highly toxic, whereas other strains have probiotic properties [81].
Another report [82] highlighted the importance of confirming strain identities thus avoiding casual links between probiotic microorganisms and strains isolated from immune-suppressed hosts. This report also proved that the cholangitis due to Bacillus in a French hospital was not caused by a probiotic.

Specific Strains with Probiotic Properties in the Genus Escherichia
Although the genus Escherichia, which belongs to the Gram negative family Enterobacteriaceae, is mainly known for its severely virulent serotypes (e.g., E. coli O157:H7), Escherichia coli is a very common inhabitant of the lower intestine and even a probiotic strain is known: Escherichia coli Nissle 1917 (EcN). As mentioned before Escherichia coli Nissle 1917 together with other probiotics have been proven to treat constipation [43] and inflammatory bowel disease [83]. This strain could also relieve gastrointestinal disorder, ulcerative colitis, Crohn's disease [84], even colon cancer [83], however more research is necessary.

Claimed Health Benefits of Individual Probiotic Microorganisms
Many publications using well-designed and well-conducted trials substantiate the health benefits of specific strains of probiotics on the risk reduction and management of a variety of diseases and conditions. Some of the documented health claims of probiotics proposed by their authors include: stimulation of various components of the immune system, gut immune response and intestinal homeostasis [85]; prevention and treatment of diarrhoea [33,39,42,86]; improvement of faecal properties and microbiota, treatment of irritable bowel syndrome, inflammatory bowel disease and constipation [43,[59][60][61]85,86]; prevention and treatment of Clostridium difficile-associated diarrhoea in adults and children [62,87]; alleviation of symptoms of lactose intolerance and other food allergies [86]; prevention of necrotising enterocolitis in preterm infants [88][89][90]; decrease in plasma cholesterol level [35,85,91]; improvement of Helicobacter pylori eradication regimens [92]; therapeutic effects by supporting the immune response of HIV-infected children and adults [93,94], anti-proliferative activity on tumour cells [95,96]; reduction of viral-associated pulmonary damage through controlling immune-coagulative responses and clearing respiratory viruses [37]; immune-stimulatory properties of low molecular mass molecules produced by probiotic bacteria [97]. Although probiotics have even been proposed as treatment for eczema [98], randomized controlled trials to date do not have sufficient evidence to recommend probiotics as primary prevention [99,100]. It is important to add that much more must be done to identify scientifically proven mechanisms of action with translational pre-clinical safety studies as well as rigorous clinical trials. To prove a health claim sample sizes of trials must be sufficient, heterogeneity of study designs (targeted population) must be enabled and accurate identification of strains should be conducted.
Also, several animal studies have observed positive effects of probiotic administration [101][102][103][104] which can have a consequential positive effect on public health of humans by lowering antibiotic consumption for animals used in food production. This will consequently lower the presence of drugs and multi-resistant organisms in the environment (including drinking water). Studies on animals are also the basis for in vivo human trials resulting in complex knowledge. However, caution should be applied, as successful use of probiotics in animal studies does not necessarily mean that similar protocols will be successful for human trials, especially for patients. Such, was the case in the study on the use of probiotics in patients suffering severe acute pancreatitis where a significantly higher mortality was observed for the probiotics group than for the placebo group [105], although these results were unexpected in the light of the results of animal studies [106]. However, this case is not representative as normally a probiotic would not be administered to this category of patients with multi-organ failure. It was later postulated that an error in the treatment, which led to the adverse cascade of events that caused organ failure and ultimately death, was perhaps due to the high incidence of gut ischemia in the treatment group [107].
The most common probiotic microorganisms with claimed health benefits for humans from the most recent scientific literature are noted in Table 1. Where data was available, strain specific data was added in brackets before the reference.

Genus Species
Recently published health claims with references (strain specific date is noted where available)
Some of the health claims of individual probiotic microorganisms noted in Table 1 are based on in vitro using cell cultures or animal models and others are based on in vivo studies. From the table we can see that health claims are very diverse and range from managing various gastrointestinal diseases or disorders to exhibiting antibiotic properties and reducing total cholesterol. Some authors even claim that probiotics reduce tumour growth, modulate brain activities and reduce allergies. However, many authors stress in their conclusions that more randomized double-blinded trials need to be conducted in the future before making recommendations.

Conclusions
This article shows that the research and subsequent knowledge on microorganisms with probiotic properties is by far not complete and we are probably only reaching the tip of the iceberg as there are many different strains of microorganisms with diverse health benefits.
The literature overview shows that, although the vast majority of probiotics are generally regarded as safe (GRAS) and beneficial for healthy individuals, caution is needed in selecting and monitoring of probiotics when administering probiotics to patients with compromised immune systems, leaky gut or critical illnesses. The most common observed adverse effects include: sepsis, fungemia and gastrointestinal ischemia. Therefore, while the overwhelming existing evidence suggests that probiotics are safe, complete consideration of risk-benefit ratio before prescribing is recommended [107].
Although it is a scientific fact that certain microorganisms can kill us, the questions arise: do certain microorganisms also keep us alive? Would it be more correct to imply that the post-modern rise of conditions and diseases such as obesity, celiac disease, asthma, allergy syndromes, type 1 diabetes and Chrohn's disease; are not only due to our genome but are also a result of the adaption of gastro-intestinal microorganisms caused by lifestyle changes, excessive intake of processed foods and sugar [7,10,168]? We cannot overlook the fact that microorganisms outnumber human cells by tenfold; the majority of which are found in the human intestinal tract [3]. Microorganisms play an important role in our gut associated lymphoid tissue, thus having profound influence on our immune system [169] and beyond. A dramatic manipulation of the intestinal microbiota involving faecal microbiota transplantation has shown remarkable clinical effectiveness for recurrent Clostridium difficile infection and ongoing studies are investigating effectiveness for other diseases [170], thus again proving the complexity of our intestinal microbiota.
Although the discovery of antibiotics was an important breakthrough in the 20th century and drugs such as penicillin and streptomycin have saved millions of lives, there is always collateral damage as our commensal microbiota is also affected [10,171]. Even though the intestinal microbiota is usually not permanently modulated with probiotic administration, this does not mean that during acute disruption of the sensitive intestinal microbiota balance, transiently present probiotics do not aid the permanent microbiota in restoring this balance.
With regard to probiotics it is also important to be careful with the science and not to oversell it [168,172]. Most probiotic products at the moment do not to go through pre-market approvals and are commonly used for a much wider range of scenarios in which their efficacy is not well established [173]. Therefore, future health claims concerning probiotics and their safety will critically depend on scientific evidence through science-based clinical studies on targeted population [174]. Another important issue is correct strain identification of each probiotic, for which expanding the internationally recognised culture collections of taxonomically classified and deposited probiotics is necessary [172]. This would assure strict use only of tested strains [175] with known profiles in compliance with regulatory pathways for 'fit for human use consumption' protocols.
Perhaps it will be possible that in the future probiotics will be used as approved drugs that will be prescribed together with/or instead of antibiotics for certain conditions such as ear infections or sinusitis [10]? An interesting concluding thought of this overview is that even though it is true that certain microorganisms can make us sick and even kill us, certain microorganisms are beneficial.

Conflicts of Interest
The author declares no conflict of interest.