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Maximizing Clinical Benefits of Probiotics: Matching Metagenomics, Patient Age and Microbial Composition


December 1, 2013
by John G. Thomas, MS, PhD; Khaled S. Seifelnasr, BDS, DDS

I. INTRODUCTION: SETTING THE STAGE FOR PROBIOTICS
It started with an observation in 1928, Robert Fleming recognized the impact of penicillin with the subsequent evolution of antibiotic therapy; and it heralded the beginning of a journey which, unfortunately, has not fulfilled its promise for a variety of reasons. In the 1960s methicillin-resistant Staphylococcus aures (Gram Positive) emerged and eventually multiple resistant mechanisms addressing Gram negative rods. The ultimate destiny was defined by biofilms, where recalcitrant communities of microbes had little response to antimicrobial therapy. The CDC recently announced that 23,000 deaths annually(www.helio.com/infectious diseases) are associated with antibiotic resistance and the NY Times article (well.logs.nytimes.com 2013) focusing on oral infections causing more hospitalizations , addressed endodontic infections. It was said best by Dr. Stuart Levy, when in 2002 he so eloquently described that “we are in a battle with microbes that we could not win by the use of antimicrobials” (Table 1). He clearly addressed the need for corroboration rather than inhibition and essentially completed the circle originally described by Elie Metchnikoff 1870, when he described that health was associated with the lactic acid Bacilli and that a balanced gut flora was the origin of an increased longevity.

II. METAGENOMICS AND ANTI-KOCH: REDEFINING THE MICROBIAL LANDSCAPE WHERE PROBIOTICS WORK
Metagenomics was first utilized in microbial ecology of soil (Ecogenomics) in the mid 1990s, recognizing the limitations of routine cultures. Ninety-nine percent of microbes are unculturable, Viable but Non-Culturable (VBNC). Metagenomics was based on emerging molecular pathology, using “shot-gun DNA Analysis” via environmental gene sequencing, to produce a profile of genetic diversity in the sample. Metagenomics represented the inherent genetic power of microbes, recognizing the total number of genes and sequential expressions elaborated in each community.1,2,3,4

The utilization of metagenomics unmasked other “Omic Technologies”, including genOmics, proteOmics, transcriptOmics, and metablOmics together with bioinformatics.5 This drew a global picture of how a microbial cell operates in its community and addressed the Systems Microbiology approach featuring total activities of microbes in a community cultured and non-cultured. It also created a new vocabulary (Table 2).

Figure 1 dramatizes the differences of the 23,000 genes of Homo sapiens versus the eight million genes of the microbiota (16S rRNA Bacteria) and mycobiota (18S rRNA Fungi). This diversity could be better understood by addressing the microbial population as an organ system not individual species. Figure 2 addressed the gene pool relative to a “microbial clock” which highlights four areas in the cohabitation of the microbial population with Homo sapiens during a lifetime. It focuses on the diseases that are associated with an aging population and the first 18 months of life. These four are critical in defining microbiota and mycobiota as a fingerprint for health and/or disease. Furthermore, it became apparent that the use of metagenomics focused on microbial population shifts and “cluster analysis” emphasizing changes at the Phyla or Family level, rather than individual species.6 Of the more than 10,000 species and 100 Phylum, 10 are common in humans. Of this group, four are most associated with health or disease: 1. Acintobacteria 2. Bacteroidetes 3. Firmicutes and 4. Proteobacteria. Most recently the ratio of Firmicutes to Proteobacteria has been shown to be predictive of outcomes: >1 predicting illness and <1 predicting health. Recent data from mice studies suggests introduced commensals function only when matched with appropriate host species. These shifts could be as important as predictors and/or modulators in disease management, as the more traditional “professional pathogens” certified by Robert Koch and Louis Pasteur.

We have used “Cluster analysis” with “Phyla signature” and “Family genetics” in two separate recent studies of West Virginia University (WVU) patient plaque samples to address: 1. Dementia and 2. Oral health.

In dementia7, distinct pattern difference were noted (Fig.  3) with a decrease in Fusobacteria and an increase with Bacteroidetes. For oral health8, an atypical pattern evolved favoring Clostridiales, which has its Phylogenetic assignment with Firmicutes. In the ” Enterotypes of Human Gut Microbes”9 an international collaborative study published in 2011, humans were classified into three types (1, 2, or 3) based on cluster analysis of stool microbiota. The implication was that this could be as important as blood group antigens for transfusion, when matching microbial interventions.

III. TARGETS FOR PROBIOTICS: CHALLENGES AND OPPORTUNITIES
Table 3 shows the growing lists of acute and chronic diseases that have been associated with unbalanced flora and antibiotic caused overgrowth. NEC (necrotizing enterocolitis in the newborn), obesity, autism, cancer, dementia and diabetes have all been identified with an abnormal or large scale shift in the commensal populations, called dysbiosis. Most of these have addressed the GI tract (Table 3A) and the importance of this population shift being defined by signals in selective gut dendrites. Potential control of these diseases has been orchestrated in two pathways with probiotics:

1. Using the consequence of the microbial metabolism on the immunologic signals of neurotransmitters

2. Using the competition between the microbes for nutrients and the ability to alter the composition of the microbiota/mycobiota.10

In dental diseases (Table 3B), this situation is pervasive in periodontal pathology: gingivitis, halitosis, caries and fungal overgrowth, where a shift in the microbial population could be manifested in a microbial change in the composition of the dental plaque or biofilm.11,12 Since 2000 we have addressed the ICU arena of ventilator associated pneumonia (VAP) and the collateral damage of dental plaque on reduced airway due to endotracheal occlusion.13,14 These also can be associated with the Microbial Clock and should be recognized as part of the continuum between the microbiota/mycobiota of the oral cavity and the microbiota/mycobiota of the GI tract. It is after all, a continuum, although the organism population is different in its diversity and quantity.

IV. PROBIOTICS: EVIDENCE BASED DENTISTRY
The use of prebiotic/probiotics in dentistry has evolved over the last 25 years highlighting their use from adjunctive care to a therapy strategy.15,16 Recently the use has addressed the metagenomics of the oral cavity and parallels the impact of a balanced microbial gastrointestinal (GI) microbiota17; it is after all a continuous anatomy.

There are well over one thousand published articles since 2000 on the use of pre and probiotics.16,18 Most have focused on Lactobacillus and Bifidobacterium sp, addressing salivary mutans as a consistent biomarker of bioburden reduction (Table 4).

Here we focus on significant review articles that consolidate scientific evidence. In 2010, JJ Reddy and Associates18 summarized a significant amount of probiotic information addressing its potential use in the entire dental clinical practice range. Goldstein et al addressed the lack in continuity of manufactures’ protocols where CFU dose at site of manufacture often did not match activity at time of purchase.19 In 2005 Meurman15 addressed the role of probiotics, recognizing that there was a need for greater information, and that there was a recognized focus on two genera, Lactobacillus and Bifidobacterium (Complications with Candida albicans providing a recent impact).

All three highlighted an international origin with a European and Scandinavian focus. In 2010 Williams17 stressed the probiotic pharmacologic arm, addressing unit of inoculums, and ingredients within the label that would affect the outcome, and stressed the need for a double-blind randomized placebo control trial. She stressed that using a cocktail/synergistic pool of microbes could have a benefit based on administration and frequency of use. In 2010 H. Singh Grove and F. Luthra12 described in the review “Probiotics, the Nano Soldiers of Oral Health”, the untapped potential of probiotics. He reinforced the work of previous investigators addressing the potential value, emphasizing that the safety signature was good and that there were unique options in addressing application. He addressed the differences between probiotic therapy and replacement therapy and the importance of strain identification.

In 2012 and 2013 the theme expanded to include the oral microbiome in health and disease. M. Zarco et al11 addressed the utilization of the oral cavity as a reflection the microbiome status; this study emphasized the biofilm and relative portions of the microbes within the oral cavity. A major feature was the discussion that possible gingival inflammation may modulate systemic disease and the impact of probiotic treatment as an immunologic modulation. Finally, Paton et al20 addressed the larger scale of bio-engineered or microbioming microbes in oral cancers, addressing conventional oral or gastrointestinal probiotic strategies. Their approach was associated with modified microbes that could share features of probiotics and pre-biotic administration.

V. PROBIOTICS: OLDEST FORM OF THERAPEUTIC INTERVENTION
As established by the World Health Organization in 2002, the definition for probiotics includes the following: Living microorganisms, a benefit when administered in adequate amounts, non-toxic, usually administered with a prebiotic. A prebiotic is defined as a functional food ingredient which was non-digestible with beneficial effects manifested through the metabolism of the probiotic and is addressed as complementary to probiotics. The history of probiotics is in itself an interesting saga. It involves a Ukrainian microbiologist, born in 1845, who received the Nobel Prize in 1905 for the theory that lactic acid could enhance longevity by preventing putrefaction in the gut.

Utilization of probiotics has been extraordinary and universal, as is shown in Figure 4; various regions within the world have addressed the use of probiotics/prebiotics as appropriate for the diseases and the clinical presentations within those regions. In the US (with latest statistics of 2010), the value was 11.3 billion dollars in sales and expected to reach 15 billion by the end of 2013; overall, 30 percent of the US population uses probiotics. The global expenditure of 85.7 billion dollars is anticipated to increase by 15 percent per year.

WHO Regions highly involved with probiotic use include: the US—region 1, Scandinavia and European areas—region 2; and Japan/Asia Pacific— regions four and five (which have the fastest growing GI use of probiotics). It is important to recognize that in 2008 there were 243 new probiotics/prebiotics products on the market with 2009 seeing essentially double that number.

The Mechanisms of Action (MOA) of probiotics are organ and site specific and multifactorial, depending upon the individual organism, its selective pressure, and the environment in which it is utilized. Table 5 describes the most recent evolution of these activities, defining their pharmacological and clinical application versus the microbiologic or anti-biofilm application. The MOA for an individual organism (the lactobacillus cell) is described in Figure 5. By highlighting the diversity with which a single organism may selectively address its metagenomic landscape, one may tailor a unique cocktail. Mixed options are available for up to 15 organisms.

Table 6 lists the most commonly used antimicrobial strains and highlights the importance of Lactobacillus genera and Bifidobacterium genera. Other genera are far less frequent, although they may be specific for particular oral and/or systemic diseases. These features highlight the need for multiple options (Table 6) in selecting patient populations paralleling the selection of antibiotics. Questions to address include: the number of different organism strains in the probiotic mixture, how it is administered, the frequency of its administration, and the length of time for which it is used. Other questions are: What is the mechanism of action? What is the target disease and means by which it promotes health of the existing metagenomics? Are there any established side effects? What are the consequences if it is administered concurrently with antimicrobials? Several investigations highlighting these variables are listed for periodontics21, oral pathogens22, gingivitis23, oral streptococci24, periodontal pocket recolonization25 and caries prevention.26

In evaluating these questions, it is important to recognize and highlight the growing use of the Sacchromyces boulardii. This is a non-human colonizing yeast resistant to antimicrobial therapy, and because of its size and biphasic nature, it is the universal co-aggregate that helps build a multispecies matrix.

Given the daunting task of matching a variety of clinical and microbiologic parameters with a plethora of probiotics, we have developed a comprehensive probiotic database for healthcare professionals that will provide consistent reliable treatment planning options, while also providing continuing evaluation of the published literature. Our goal is to provide healthcare providers easy access to information regarding probiotics while providing new research tools.

The searchable database contains several search functions: age, disease, surgical procedures, probiotics, brand/microbes/drug grade, drug interactions, and contraindication. We have titled this BAC-2-HEALTH (Table 8). It is available under the West Virginia University SOD website http://dentistry.hsc.wvu.edu/Oral-Health-Thomas/ or Http://dental.HSC.WVU.edu/Oral-Health-Thomas/Home and complements other research tools and educational resources. These include detailed descriptions of selected topics as well as fifteen 30 minute mini-educational topics.

It is our contention that one of the most misunderstood features of probiotic selection is the impact upon resident flora. Eighty percent of the microbes are non-transient and associated with age, particularly adult and geriatric populations in the “Microbial Clock”. Further, it is the goal of the database to provide information about the quality of the probiotics recognizing there is significant variability in the Colony Forming Units (CFU’s) within the probiotic, the storage requirement, the impact of the improper storage, and a plethora of problems associated with manufacturing.

To promote awareness of probiotics, the need for continuing education and updating of the evolving management opportunities, we have structured an online survey. This survey has been administered over the last three years both nationally and internationally, to a variety of individuals of various educational backgrounds including dental students, dental hygienists, dentists, and specialists in such different places as Germany, Cardiff Wales, Australia, New Zealand, and Japan. The results are intriguing with evaluations based on the six WHO regions and highlight the need for the BAC-2-HEALTH website. It is our goal to summarize the survey and compare it to other results internationally and integrate the studies and observations into the BAC-2-HEALTH website for database analysis. We encourage you to take the survey as we will publish the results in an upcoming edition of this journal.

VI. APPLICATION: T-COC
We introduced the concept of “Tailored-Comprehensive Oral Care” (T-COC) in 2007 whi
ch integrates the universal concept of Minimal Intervention (MI). It also incorporates patient empowerment. This concept is highlighted in Figure 6, which describes a five part scheme focusing on traditional aspects of oral health and includes the use of probiotics with patient compliance. One scheme uses an aseptic mouth rinse one hour prior to the application of the probiotic which is maintained during sleeping hours. As with all patient care, it is important to recognize that this is dynamic and needs continual reassessment. The key words are “tailoring” and “matching” , and recognizing the limitation of probiotics as outlined in Table 9. No single probiotic meets the needs of all patients. General guidelines should include critical evaluation based on data and published studies highlighting strain specificity. Probiotics may require 109 to 1011 viable cells per day and the prebiotic 20 to 30 grams per day. Continued intake may be required to maintain benefits and the prebiotic may be an excellent option to modify oral or gut flora on a long term basis. It is important to recognize that probiotic colonization is temporary, not establishing host residence or biofilm integrity.

VII. SUMMARY: “WE LIVE IN A MICROBIAL WORLD”
We live in a “microbial world” (mostly biofilm), emphasizes our growing recognition that to survive requires harmony with our microbial cohabitant. Recent clarification of these cohabitants by metagenomics gives us a chance to establish a healthy microbiota as a pre-eminent feature of dental management, addressing Minimal Intervention. The cost of oral disease and its potentially associated systemic links may be up $96 billion per year (Fig. 7). Elucidating the microbiota and mycobiota gives a chance to fulfill the promise that biomining and stewardship of the gene pool (harbored by the total microbial community), is our best approach to disease management.

One means of reestablishing this stewardship is the utilization of probiotics, recognized as a theme in the 1870s by Elie Metchnikoff. Probiotics traditionally have been viewed as complementary intervention, but today that theme is changing dramatically. The use of probiotics is now viewed as a therapeutic strategy and not simply as adjunctive care. This very promising strategy must also take into account the microbial landscape and gene diversity (Metagenomics), the patients Microbial Clock and proper probiotic composition based on scientific evidence. Perhaps most underestimated is matching pre/probiotic selection with an Enterotype classifying the stool microbial landscape, defining the patient metagenomic signature. OH


John G Thomas, MS, PhD, Clinical Professor Department of Endodontics, WVU SOD and Professor Emeritus Department of Pathology, WVU SOM

Khaled S Seifelnasr, BDS, DDS, Department of Endodontics, WVU SOD Morgantown, WV, USA

Acknowledgements: Ariel Mooney, RDH, BS, Colleen Conlon, BS, BA, MS, Nicole Andreini, BS, 3rd Year Dental Students WVU SOD

Oral Health welcomes this original article.

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