July 2016 Ancient Dental Calculus Subgingival Microbiome Shifts Caries Pathology Uncultivated Oral Bacteria JournaCALIFORNIA DENTAL ASSOCIATION

THE ORAL MMICROBIOME:ICROBIOME: Critical for Understanding Oral Health and Disease Floyd E. Dewhirst, DDS, PhD You are not a statistic.

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DEPARTMENTS 397 The Editor/Dentistry as an Endurance Sport

399 Letters

401 Impressions

459 RM Matters/Preservation of Property: A Critical Obligation

463 Regulatory Compliance/A Patient’s Right to Access Records Q-and-A 401401 467 Ethics/Undertreatment, an Ethical Issue

469 Tech Trends

472 Dr. Bob/A Dentist’s Guide to Fitness

FEATURES 409 The Oral Microbiome: Critical for Understanding Oral Health and Disease An introduction to the issue. Floyd E. Dewhirst, DDS, PhD

411 Dental Calculus and the Evolution of the Human Oral Microbiome This article reviews recent advancements in ancient dental calculus research and emerging insights into the evolution and ecology of the human oral microbiome. Christina Warinner, PhD

421 Subgingival Microbiome Shifts and Community Dynamics in Periodontal Diseases This article describes shifts in the subgingival microbiome in periodontal diseases. Patricia I. Diaz, DDS, PhD; Anilei Hoare, PhD; and Bo-Young Hong, PhD

437 Understanding Caries From the Oral Microbiome Perspective This article discusses how microbiological cultivation, molecular identification, gene expression and metabolomic analyses show the importance of the entire microbial community in understanding the role of the microbiome in the pathology of caries. Anne C.R. Tanner, BDS, PhD; Christine A. Kressirer, PhD; and Lina L. Faller, PhD

447 Uncultured Members of the Oral Microbiome The aim of this review is to list the uncultivated members of the oral microbiome, discuss the reasons why some bacteria are difficult to culture in vitro and describe recent advances in culturing previously uncultivated oral bacteria. William Wade, BSc, PhD; Hayley Thompson, BSc, PhD; Alexandra Rybalka, BSc, PhD; and Sonia Vartoukian, BDS, FDS, PhD

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Volume 44, Number 7 JournaCALIFORNIA DENTAL ASSOCIATION July 2016 CDA Classifieds.

Free postings. published by the Editorial Upcoming Topics Letters to the Editor California Kerry K. Carney, DDS, CDE August/Student Research www.editorialmanager. EDITOR-IN-CHIEF Priceless results. Dental Association September/Pathology com/jcaldentassoc 1201 K St., 14th Floor [email protected] October/Sugar Sacramento, CA 95814 Ruchi K. Sahota, DDS, CDE Subscriptions 800.232.7645 ASSOCIATE EDITOR Subscriptions are available cda.org Advertising only to active members of Brian K. Shue, DDS, CDE Doug Brown the Association. The ADVERTISING SALES CDA Offi cers ASSOCIATE EDITOR subscription rate is $18 and [email protected] is included in membership Kenneth G. Wallis, DDS 916.554.7312 PRESIDENT Floyd E. Dewhirst, DDS, PhD dues. Nonmembers can GUEST EDITOR [email protected] view the publication online Permission and at cda.org/journal. Clelan G. Ehrler, DDS Andrea LaMattina Reprints PRESIDENT-ELECT SENIOR PUBLICATIONS Manage your subscription SPECIALIST Andrea LaMattina online: go to cda.org, log in [email protected] SENIOR PUBLICATIONS and update any changes to SPECIALIST Blake Ellington your mailing information. Natasha A. Lee, DDS [email protected] VICE PRESIDENT TECH TRENDS EDITOR Email questions or other 916.554.5950 [email protected] changes to membership@ Courtney Grant cda.org. COMMUNICATIONS R. Del Brunner, DDS Manuscript SPECIALIST SECRETARY Submissions [email protected] Jack F. Conley, DDS www.editorialmanager. EDITOR EMERITUS com/jcaldentassoc Kevin M. Keating, DDS, MS TREASURER [email protected] Robert E. Horseman, DDS CDA classifiedsclassifieds wworkork harder to HUMORIST EMERITUS Stay Connected cda.org/journal bbringring you resuresults.lts. SeSellinglling a practice Craig S. Yarborough, DDS, MBA Production or a piece ooff equipment? Now you SPEAKER OF THE HOUSE [email protected] Val B. Mina can include photos to help buyers SENIOR GRAPHIC DESIGNER Walter G. Weber, DDS Go Digital cda.org/apps see the potential. IMMEDIATE PAST PRESIDENT Randi Taylor SENIOR GRAPHIC DESIGNER Look for this symbol, noting additional video [email protected] content in the ePub version of the Journal. And if you’re hiring, candidates anywhere can apply right from Management Peter A. DuBois the site. Looking for a job? You can EXECUTIVE DIRECTOR Journal of the California Dental Association (ISSN 1043–2256) is published monthly by the post that, too. And the best part— Jennifer George California Dental Association, 1201 K St., 14th Floor, Sacramento, CA 95814, 916.554.5950. it’s free to all CDA members. CHIEF MARKETING OFFICER Periodicals postage paid at Sacramento, Calif. Postmaster: Send address changes to Journal of the California Dental Association, P.O. Box 13749, Sacramento, CA 95853. Cathy Mudge The California Dental Association holds the copyright for all articles and artwork published All of these features are designed to VICE PRESIDENT herein. The Journal of the California Dental Association is published under the supervision of PUBLIC AFFAIRS help you get the results you need, CDA’s editorial staff . Neither the editorial staff , the editor, nor the association are responsible for any expression of opinion or statement of fact, all of which are published solely on the authority faster than ever. Check it out for Alicia Malaby of the author whose name is indicated. The association reserves the right to illustrate, reduce, COMMUNICATIONS revise or reject any manuscript submitted. Articles are considered for publication on condition yourself at cda.org/classifieds. DIRECTOR that they are contributed solely to the Journal. Copyright 2016 by the California Dental Association. All rights reserved.

396 JULY 2016 Editor CDA JOURNAL, VOL 44, Nº7

Dentistry as an Endurance Sport Kerry K. Carney, DDS, CDE

hat if we thought of our professional lives as an endurance sport When I was in dental school, one of our instead of a business clinical instructors advised us to never run our career? What if the Wgoal was to stay mentally and physically practices continuously at 80 miles an hour. strong and healthy? What if our primary driver was to remain happy and to continue to enjoy helping our patients achieve and maintain oral health over the decades that veins, now it is hemorrhoids. We all have nutritional guidelines for dentist athletes? span the course of a career in dentistry? taken ergonomic classes that reinforce Maybe we should have spent some time Many practice management courses the need for good posture and healthy studying what we should be eating. What spend a lot of time trying various strategies body mechanics. However, it only takes individualized diet would provide us the to maximize production: the shortest the restoration of the distal aspect of appropriate quantity and quality of calories possible time to return on investment, an upper third molar in a patient with for our level of activity? When should we the most effi cient scheduling to meet restricted opening to know that the dentist be eating and what should we be eating production goals, the minimizing of will assume whatever contorted position to stay alert and relaxed? Caffeinated nonproductive downtime. If the offi ce is necessary to complete the procedure. afternoon pick-me-ups are probably not be open, it should be producing at maximal We buy the aids: head-mounted the best answer for endurance training. potential. Time is money and overhead is lights, stools with armrests and loupes Every sport has a psychological high, so increasing speed and profi ciency for improved vision, but if dentistry aspect. Psychology for the dentist athlete is an obvious goal in order to achieve is an endurance sport, we need to be is complicated. The dentist needs the success. If monetary success is the measure thinking about how to help the body ability to focus on the fi ne motor skills then we run, run, run (or, as my husband strengthen the muscles that are not for performing surgical and therapeutic likes to say, “Work, work only!”). engaged in that habitual position. We interventions but everyday dentistry When I was in dental school, one of need to learn how to stretch and lengthen requires a lot more than that. The dentist our clinical instructors advised us to never those muscles that are always fl exed in needs the kind of team training to be run our practices continuously at 80 miles the dentist’s habitual work position. able to work smoothly and effi ciently an hour. He said we would need to be able In dental school, we spend a lot of with offi ce staff and colleagues. to accelerate our speed when necessary time on head and neck anatomy, but As dentist athletes, we have the added but we needed to maintain a sustainable recently I have had to spend a lot of complication of patient interaction to pace, one that would not wear us out. I time understanding the workings of the consider in our mental training. In order to was reminded of that advice when I was muscles of the back, legs and body core. be able to endure and be happy, we have doing some physical therapy recently. I have learned that the health of the to learn how to compartmentalize some For a career in dentistry to be psoas muscle is critical in those who, like aspects of our interactions with patients experienced as an endurance sport, we dentists, sit for a living. If dentistry is and staff. Our patients trust us to care need to look at all the pieces that go into an endurance sport, we should dedicate for them. We must be able to empathize that sport and how we can train to excel time to understanding our muscular- with our patients. Even when our patients in that sport. A day in the offi ce requires skeletal interactions and train to keep are transported to us on vehicles of pain physical fi tness. Dentistry has habitual our bodies fi t and ready for the fatiguing, and fear, we cannot get wound up in positions that can lead to stress on some straining rigors of everyday practice. psychodramas of their own making. parts of our bodies and weaken others. The nutritional aspect of training for Some people are natural athletes and In the fi rst half of the century, the an endurance sport has to be considered some are natural psychosocial athletes. dentist’s habitual position was standing, as well. In dental school, we learned For some of us, the psychological aspects now the seated position is usually preferred. basics about nutrition and its effects of dentistry are what trip us up, burn us An occupational hazard then was varicose on the dentition. But what about the out and cause us to have a short and/

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or unhappy career in dentistry. This psychosocial component of training is frequently undervalued, overlooked and underdeveloped. We need to train for a mental fi tness that can sustain us along the emotional and psychological obstacle course that must be negotiated day after day. Every training program has to incorporate rest and relaxation and dentistry as an endurance sport is no different. It is too easy to think we are indispensable and irreplaceable. The dentist athlete needs to build into the training schedule time to sleep, rest, relax and recreate. A career in dentistry is not a sprint. Dentistry is surely an endurance sport and one that is practiced not in loneliness, like the long-distance runner, but with companions and friends. It is a veritable steeplechase day after day. With some luck and effective training, it is an endurance With us, success is part sport we should be able to look forward to of the treatment plan. enjoying every day for the long haul. ■ The Journal welcomes letters With Fremont Bank, you have access to We reserve the right to edit all the financial resources you need to take communications. Letters should discuss an item your practice to a higher level. published in the Journal within the last two months or matters of general interest to our readership. Letters must be no more than 500 words and cite no more than fi ve references. With local decision making and unprecedented access to our executive team, Fremont Bank is No illustrations will be accepted. Letters well-positioned to provide you with the products should be submitted at editorialmanager.com/ and services your dental practice requires with the jcaldentassoc. By sending the letter, the author personal touch of a trusted advisor. certifi es that neither the letter nor one with substantially similar content under the writer’s Experience the Fremont Bank difference for yourself. authorship has been published or is being Call 1-888-460-6564 to start a relationship today. considered for publication elsewhere, and the author acknowledges and agrees that the letter and all rights with regard to the letter become the property of CDA. Success Through Partnership • www.FremontBank.com

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398 JULY 2016 Letters CDA JOURNAL, VOL 44, Nº7

Mental Health Dialogue

I recently had the opportunity to are specifi c personality traits needed to April 2016 Quality Improvement in glance through the April 2016 issue of the enter into the fi eld of dentistry. The fi eld Pediatric Oral Health Dental Quality Measurement Dental Quality Alliance Journal. As a licensed psychologist who has seems to demand a heightened level of JournaCALIFORNIA DENTAL ASSOCIATION worked with dentists for a number of years, attention to detail, and for some, this can I was particularly interested in the student reach the level of clinical signifi cance editorial by Christian Piers titled “A Light described in perfectionism. Dentistry

0 ADVANCING for Dark Places: Mental Wellness in Dental also appears to require a heightened MEASUREMENT AND QUALITY IMPROVEMENT IN 1 DENTISTRY School.” It seems to me that Mr. Piers level of competitiveness to succeed in James J. Crall, DDS, ScD courageously attempted to open a dialogue training and private practice. And, at 2 about mental health and suicide for dental its zenith, heightened competitiveness 3 students. He, in my opinion, correctly can lead to social isolation. Couple 4 points out that “suicide is complicated.” these traits with the rigors of dental 5 6 While his editorial indicates that school or managing a private practice 7 there have been several research studies and it is conceivable that some may 8 attempting to establish some connection become periodically overwhelmed. 9 between an elevated suicide rate and I share support with the editorial dentists, there is also a substantial body regarding self-care and access to mental of work indicating no strong correlation health services. Additionally, there may can be made based on the existing data. be a need to address any stigma that may Part of the helpful solution is the For the most part, this is due to issues be associated with an individual seeking CDA Well-Being program that was of statistical sample size in those studies these services. Finally, I would assert that wisely set up years ago. The local that support this idea. The prevailing if my anecdotal fi ndings can be supported dental societies assist the Well-Being theory seems to be that dentists are through appropriate research, this might Committee in action. Dental school exposed to high levels of stress leading prove to be an invaluable aid for dental is diffi cult; dental practice is stressful. to increased levels of depression. schools. Having this information would The Well-Being program aids people At one point in my practice, I came enable them to identify individuals who who need extra help in dealing with to realize that approximately one-third may be prone to the overwhelming sense stress. It is CDA’s helping arm where of my clients were dentists, leading me of helplessness and/or hopelessness leading confi dentially colleagues with history to contact a friend who is also a dentist. to suicide, thereby having an opportunity and/or experience in this area provide His lack of surprise when I presented to intervene prior to any harmful guidance and help to those calling this fact surprised me! He was unable action being taken by the individual. CDA’s help line (for regional numbers, to identify a possible reason other than DON J . TALLEY, PHD, NCC go to cda.org/Portals/0/pdfs/cda_ commenting that he believed dentistry Santa Rosa, Calif. wellbeing_brochure.pdf). Any number of attracted a certain type of individual and issues, including chemical dependency, that the “profession is hard on people, CDA Well-Being depression, sexual, fi nancial and eating physically and mentally.” Over my years With great interest I read in the disorders, can be addressed. There are of working clinically with dentists, there April 2016 issue of Journal Christian many helpful resources to whom we has not been a single case of one of my Piers’ student editorial dealing with refer those in need. As a Well-Being dentist client’s presenting complaint traumatic and tragic loss. His writing was Committee member in San Diego for being depression. Indeed, following so provocative that I had to read it in its many years, former committee chair my routine initial intake assessment entirety in spite of students and other and CDA regional director, I assure you I have yet to determine a primary obligations calling me. I am more than the rewards we experience in seeing the diagnosis of depression for a dentist. pleased to read his perspective on a subject miracle of a colleague getting better and I do see some common themes in dentists often avoid discussing. Opening up healing is intense, even emotionally dentists with whom I work. A review the topic is much healthier than burying impactful at times, when a colleague’s life of the psychology literature in dentistry it and avoiding it. How sad to learn of the is saved. The value of CDA Well-Being supports my anecdotal fi ndings. There tragic loss of this beautiful student leader. in assistance given cannot be measured.

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Thank you, Mr. Piers, for the and my own experience, I would like and enticing their patients with heavy excellent and well-written way you to venture some possible reasons. As advertisements. Unfortunately, such dental handled a diffi cult topic. said in the article, California has many treatments frequently CAUSE problems. I commend and admire you. You will more dentists per 100,000 population During my 10 years of working for public do very well and will be an asset to our than the national average, which means clinics, I often saw previously insured profession wherever you give your talents. competition among California dentists patients come in with serious problems RONALD E . FRITZ, DDS is fi erce. In the meantime, Medicaid caused by elective cosmetic treatments Rancho Santa Fe, Calif. (Medi-Cal in California) pays among the (these patients had been dropped by their lowest reimbursements in California. Both dentists because they lost their insurance). Dental Problems factors lead the majority of California Now, I own a private offi ce in a low-income I have read the editorial in the May dentists to focus on treating the wealthy neighborhood, treating 80 percent Medi- issue of the Journal entitled “A Meaningful and/or well-insured patients, who are Cal patients. Receiving $40 for a fi lling and Glance at California’s Oral Health Care actually a minority compared to the $41 for a wisdom tooth extraction while System.” It is curious that California’s vast numbers of low-income and welfare trying to operate a “for-profi t” business, I high-income adults have more dental patients. Consequently, these dentists understand the fi nancial strain just too well. problems than the poor. Based on the tend to perform expensive, aggressive ANNA DONG, DDS information provided in the article and unnecessary treatments, persuading Pittsburg, Calif.

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400 JULY 2016 Impressionsp CDA JOURNAL, VOL 44, Nº7

Mrs. Jellyby and the Hot Rock

David W. Chambers, EdM, MBA, PhD

“Think globally, act locally.” The fi rst part of this popular phrase seems a bit vague; the second part is trivial. If your friend said he had decided to only hug the people he could actually reach, you would wonder whether he really got the concept. Perhaps the greatest send-up of this concept is Charles Dickens’s classic Bleak House. In the chapter on telescopic philanthropy, we are introduced to the Jellybys, a magnifi cently dysfunctional family of a voiceless father and an uncertain number of kids, all ill clothed, sick and infantile. One daughter is an alcoholic and another elopes to escape serving as her mother’s slave. Only Mrs. Jellyby has it together. She is the doyen of the East London Branch Aid Ramifi cation, a charitable group devoted to bringing Christianity and Western economic civility to the Borrioboola-Gha people in Africa by getting London soft hearts to donate funds for a coffee plantation and a factory to turn piano legs. Mrs. J. is the center of a network of communication that involves getting and sending hundreds of letters a day and distributing bundles of fl yers for the cause. She is a force for global change. But the oldest daughter says, “It’s disgraceful. The whole house is disgraceful. The children are disgraceful. I am disgraceful.” TheThb nub: Now for the hot rock. Imagine dropping one in the shallow end of a friend’s swimming pool. A minute or two later, you 1. All change is within reach. will fi nd that the temperature of the rock and all the water is the same. Perhaps immeasurably higher, but that certainly 2. No one changes the will not last. This is the second law of thermodynamics, system or others without being also called entropy. In any closed system that allows changed themselves. interchange, the trend is inexorably toward homogeneity. This is one of those laws that cannot be broken. But we can get 3. Change is not sustainable around it. The easy, short-term dodge is to build barriers that divide without infusions of energy from the hot from the cold, separating the haves from the have-nots. The more productive means is to make the system open — to expect and outside. encourage new energy, hope and resources from outside the system. Both accommodations have their shortcomings. Building personally profi table protective enclaves will add stress to David W. Chambers, EdM, MBA, PhD, is professor the system and it will eventually collapse. Trying harder from of dental education at the University of the Pacifi c, Arthur within must be continually maintained and will never lead A. Dugoni School of Dentistry, San Francisco, and editor to large sustainable change. The effect of the hot rock (the of the Journal of the American College of Dentists. heat coming from outside) will be greatest and most lasting on the water immediately contacting the rock. A smaller change will be caused by that hotter water that warms the water immediately next to it, but cools some in the process. The water at the deep end of the pool remains unaffected. The most effective counselors for Alcoholics Anonymous, domestic abuse, drug addiction, community development and good oral hygiene are those who live close enough to touch them. ■

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Robotic Surgical Approach for Cleft Palate Surgery A recent study aimed to assess the safety and feasibility of transoral robotic surgery for soft palate muscle reconstruction and was deemed safe for cleft palate surgery by the authors. The researchers describe using the da Vinci Surgical System in cleft palate surgery from successful testing on a cadaver to performing surgery on 10 live patients, aged 9 to 12 months. In order to measure how the robotics altered the surgical performance, a control group of 30 patients was treated ADA Code of Ethics with the same technique using conventional, nonrobotic approaches. The authors found that, on average, using the da Vinci System Celebrates 150 Years took 35 minutes longer than manual instruments, but noted that This year, the American Dental the use of the surgical robot on a cadaver provided great dexterity Association’s Code of Ethics is and excellent 3-D depth perception, which helped increase celebrating its 150-year anniversary. precision and decreased hand tremors. The authors reported that As a condition of ADA membership, they encountered zero infections and no other surgery-related members voluntarily agree to complications, which resulted in a shorter recovery time for patients. abide by the principles of ethics “We believe that the precise dissection of the palatal muscles provided by and code of professional conduct the robotic system might reduce damage to the vascularization and innervation detailed in the ADA Code of Ethics, of these muscles, as well as damage to the mucosal surfaces that could cause committing themselves to high ethical fi stula formation. In addition, this technique might improve palatal function standards of conduct and a “patient- first promise.” With a consistent and Eustachian tube function in cleft palate patients,” the authors concluded. fundamental purpose of putting the For more, see the full text published welfare of patients first, the ADA in The Cleft Palate – Craniofacial Journal, Code of Ethics is, in effect, a written 2016, vol. 53, no. 3, pp. 326-331. expression of the obligations arising from the implied contract between Intraoperative view of the left levator veli the dental profession and society. palatini muscle dissection from the nasal mucosal layer. Reprinted with permission The patient-first promise from The Cleft Palate – Craniofacial means ADA members promise to Journal, Allen Press Publishing Services. “respect,” “do no harm,” “do good,” “be fair” and “be truthful.” The ADA Code has three main components: The Principles of Ethics, the Code of Professional Conduct and required or prohibited. The Advisory While the ADA Code turns 150 the Advisory Opinions. The Principles Opinions are interpretations that apply this year, it is an evolving document of Ethics are the aspirational goals of the Code of Professional Conduct and by its very nature cannot be a the profession. The five fundamental to specific fact situations. They are complete articulation of all ethical principles consist of “patient autonomy, adopted by the ADA’s Council on obligations. The ADA Code is the nonmaleficence, beneficence, justice Ethics, Bylaws and Judicial Affairs to result of an ongoing dialogue between and veracity.” The Code of Professional provide guidance to the membership the dental profession and society, and as Conduct, a product of the ADA’s on how the Council might interpret such, is subject to continuous review. legislative system, is an expression of the Code of Professional Conduct For more, see the ADA Code at specific types of conduct that are either in a disciplinary proceeding. ada.org/ethics.

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Dental Prescribing of Antibiotics on the Rise New research analyzing data on based prescription database. According to outpatient prescriptions from 1996 to the report, the researchers’ analyses used 2013 has found a 62.2 percent increase Anatomical Therapeutic Classifi cation in the rate of prescribing of antibiotics standard codes and defi ned daily dose by dentists, according to a study in The (DDD) values. The authors normalized Journal of the American Dental Association. prescribing rates to the population For their study, the authors obtained and expressed the rates in DDDs per anonymized, line-listed data on outpatient 1,000 inhabitants per day (DID). prescriptions from a centralized, population- From 1996 to 2013, while overall

antibiotic use declined from 18.24 DID to 15.91 DID, and physician prescribing declined from 17.25 DID to 14.11 DID, Cancer Gene Spectrin May Predict Survival dental prescribing increased from 0.98 Outcomes of Mouth Cancers DID to 1.59 DID (62.2 percent), and its proportionate contribution increased from Loyola researchers have identifi ed a tumor gene that may help to predict 6.7 percent to 11.3 percent of antibiotic survival outcomes in patients with cancer of the mouth and tongue. If the gene prescriptions. The rate of prescribing spectrin (involved in the formation of cell membranes) is expressed, patients are increased the most for dental patients 4.6 times more likely to die at any given time, according to a study by researchers 60 years or older, the authors wrote. at Loyola Medicine and Loyola University Chicago Stritch School of Medicine. “Communication from dentists in If the cancer gene is expressed, a patient may require more aggressive Canada and the United States identifi ed treatment, such as radiation and possibly chemotherapy in addition to surgery. the following explanatory themes: Conversely, if the gene is unexpressed, the patient might be able to safely forgo unnecessary prescriptions for periapical aggressive treatment and undergo surgery alone. abscess and irreversible pulpitis; increased Using the Gene Expression Barcode, researchers examined publically prescribing associated with dental implants available genetic data from 54 tumor samples. The samples were taken from and their complications; slow adoption patients who had HPV-negative squamous cell carcinoma in the mouth. of guidelines calling for less perioperative antibiotic coverage for patients with Even when researchers controlled for cancer stage and other factors, patients valvular heart disease and prosthetic with the expressed spectrin gene still were signifi cantly more likely to die than joints; emphasis on cosmetic practices those in which the gene was turned off . This fi nding suggests that the spectrin reducing the surgical skill set of average gene may provide more information about survival than cancer stage alone. dentists; underinsurance practices driving The authors caution that the results are preliminary and need to be validated antibiotics to be a substitute for surgery; in an independent patient group. the aging population; and more dental For more, see the study in the registrants per capita,” the authors wrote. journal Otolaryngology—Head Antibiotic prescribing should be and Neck Surgery, published reviewed to make sure that practitioners online before print April 19, 2016. are compliant with guidelines, according to the researchers, who noted that in doing so, Michael J. Zilliox, PhD, director of most practitioners can fi nd opportunities to the Loyola Genomics Facility and lab prescribe less often and for shorter durations. manager Gina Kuff el. Image courtesy For more, see the research published of Loyola University, Chicago. in The Journal of the American Dental Association, May 2016, vol. 147, issue 5, pp. 320-327.

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Techniques Improve for Determining Male or Female Jawbone Scientists recently discovered new data that improve techniques for determining whether a jawbone comes from a man or woman. The scientists Double immunofl uorescence staining of discovered that the diff erences that help to distinguish between the jawbone PDGFRβ/E-Cadherin on cryosections from oral of a male and a female are diff erent if the subject has a meso-, dolicho- or tumor biopsies. Images on top half are x10 braquifacial pattern (the three types of anthropometric profi les). magnifi cation; bottom are x60 of circled area. “These results imply that an assessment of the vertical facial pattern of the individual is required before a sexual diagnosis Novel Marker of Oral of the mandible is proposed,” the authors wrote. Cancer Discovered Scientists at the University of Granada and the National Museum of Natural Science (of the CSIC) have applied a new, For the fi rst time, researchers have more accurate technique in order to analyze identifi ed a reliable marker (PDGFRβ) the diff erences in mandible size and shape, to detect carcinoma-associated which are linked to gender. The new fi broblasts (CAFs) — which are cells technique will be useful when within the tumor that encourage determining whether a bone comes growth and metastasis — in oral cancer from a man or a woman. tissues, according to new research. For their study, the team of With this discovery, anti-PDGFRβ treatment could soon be combined with researchers analyzed the jawbones existing tumor treatments to provide of 187 adult subjects (92 men and 95 women) from Granada using lateral a more effective cancer therapy. teleradiography of the cranium. The size and shape of the jawbones were CAFs have been shown to be studied using specifi c geometric morphometric techniques. The authors reported strongly predictive of disease severity, fi nding statistically signifi cant diff erences in the size and shape of the bones but their study has been hampered by a between men and women. This sexual dimorphism can be clearly observed diffi culty in identifying reliable markers in all the patterns, both vertical and sagittal, that were analyzed, according for their isolation from tissue samples. to a news release. The male jawbone is bigger across all subgroups. Researchers identifi ed a set of For more information, see the study published online ahead of print collagen genes they expected to be in the Journal of Comparative Human Biology, Jan. 23, 2016. largely CAF-specifi c, namelyCOL1A1 , COL1A2 and COL3A1. Using a large gene expression dataset from the cancer genome atlas comprising hundreds of oral cancer samples, they then looked for additional genes the head and neck, the identifi cation characterized a list of promising whose expression best associated with of robust and reliable markers of candidate CAF markers that may be the average expression of these three these cells is necessary to further relevant to other carcinomas,” the collagen genes. In doing so, they assess their role in tumor initiation authors concluded, adding that their identifi ed several markers, including and progression,” explained Maria “novel approach provides for a fast PDGFRβ, which they confi rmed to be Trojanowska, PhD, professor of and accurate method to identify CAF CAF-specifi c using immunostaining medicine at Boston University School markers without the need for large- assays in oral carcinoma specimens. of Medicine, in a news release. scale immunostaining experiments.” “Given the known association “Overall, this study identifi ed For more, see the research of CAFs with poor prognosis in PDGFRβ as a novel marker of stromal published in the journal PLOS certain cancers, including those of activation in OSCC, and further ONE, April 29, 2016.

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Hepatitis C Is Top Infectious Disease Killer in U.S. Deaths associated with hepatitis C reported to the CDC, including HIV, reached an all-time high of 19,659 in pneumococcal disease and tuberculosis. 2014, according to new surveillance The greatest hepatitis C burden data released by the Centers for falls on baby boomers — those born Disease Control and Prevention between 1946 and 1964 — many (CDC). And, a second CDC study of whom have unknowingly been showed that annual hepatitis C-related living with the infection for many mortality in 2013 surpassed the years. According to a study published total combined number of deaths previously in The Lancet Infectious from 60 other infectious diseases Diseases, many baby boomers were infected during medical procedures in the years after World War II, when injection and blood transfusion technologies were not as safe as Governor Signs Historic Package of Bills Restricting they are today. Without diagnosis Tobacco Use and treatment, they increasingly develop liver cancer and other life- Gov. Jerry Brown has signed a package of fi ve bills that are the most threatening hepatitis C-related signifi cant set of tobacco restrictions in years. Brown signed: diseases, and they may unknowingly ■ SB 5 X2 by Sen. Mark Leno classifi es e-cigarettes as tobacco products. transmit the disease to others. This will make them subject to smoke-free laws, age restrictions and other The surveillance data also rules governing tobacco products. point to a new wave of hepatitis C ■ SB 7 X2 by Sen. Ed Hernandez and Assemblymember Jim Wood, DDS, infections among people who inject raises the age to buy tobacco products from 18 to 21. drugs. Acute cases of hepatitis C ■ AB 7 X2 by Assemblymember Mark Stone closes loopholes in the state’s infection have more than doubled smoke-free workplace laws. since 2010, increasing to 2,194 reported cases in 2014. The new ■ AB 9 X2 by Assemblymembers Tony Thurmond and Adrin Nazarian cases were predominantly among requires all schools to be tobacco-free. young,young white individuals with a ■ AB 11 X2 by Assemblymember Nazarian establishes historyhisto of injection drug use, living a tobacco licensing fee program under the state Board in rruralur and suburban areas of the of Equalization. MidwestMidw and Eastern United States. “These bills are the most signifi cant set of tobacco “Because“B hepatitis C often has few restrictions in years and we thank the governor and Legislature notinoticeablec symptoms, the number for taking this historic step,” CDA President Ken Wallis, of newne cases is likely much higher DDS, said. “Dentists see the devastating eff ects of tobacco than what is reported. Due to limited use every day and we are very pleased that California has sscreeningcree and underreporting, we taken bold steps to protect our residents from these deadly estimateestim the number of new infections products.” is closerclo to 30,000 per year,” said JohnJohn W. Ward, MD, director of California becomes the second state, after Hawaii, to raise CDC’sCDC Division of Viral Hepatitis, the smoking age to 21. More than 100 cities, including in a nnews release. “We must act San Francisco, have also passed this policy. The state now tto diagnose and treat hidden also joins a growing number of states regulating infectionsinfec before they become deadly e-cigarettes like tobacco products in order to andand tto prevent new infections.” combat the exploding use of e-cigarettes by teens. SeeSe more published in Clinical InfectiousInfect Diseases, vol. 62, issue 10, pp. 1287-1288.1287-

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The Oral Microbiome: Critical for Understanding Oral Health and Disease

Floyd E. Dewhirst, DDS, PhD

GUEST EDITOR

Floyd E. Dewhirst, DDS, ince the time of Koch These bacteria interact with each PhD , is a senior member of and Pasteur, medical other, with host tissues and the immune staff in the department of microbiologists have focused and infl ammatory response systems. microbiology at the Forsyth Institute in Cambridge, narrowly on fi nding the Investigators have realized that to Mass., and a professor specifi c pathogen that causes fully understand human health and in the department of oral Sa particular disease. Guided by Koch’s disease, we need to understand not medicine, infection and Postulates, investigators proved just the few suspected pathogens, but immunity at the Harvard causation of many diseases including rather all members of the microbiota. School of Dental Medicine in Boston. polio and diphtheria. However, not Microbiome is the term used for the Confl ict of Interest all infectious diseases are caused by a entire microbial community, including Disclosure: None reported single organism. This is particularly true pathogens, commensals and mutualists. of diseases involving biofi lms where The National Institutes of Health the community can be composed of recognized the importance of studying several hundred species. The bacteria the human microbiome and launched most associated with causing caries, the Human Microbiome Project (HMP) Streptococcus mutans, and periodontal in 2008. The microbiomes of oral, gut, disease, Porphyromonas gingivalis, are nasal, vaginal and skin body sites were not exogenous pathogens like Vibrio examined in 300 healthy individuals to cholera or Corynebacterium diphtheriae, lay a foundation for future microbiome but rather common members of the investigations. Information and oral microbiota that are present at scientifi c methods developed in the low numbers in most people without HMP are now routinely used in studies causing disease. The bacteria in the of the oral cavity and oral diseases. In oral cavity include those associated this issue of the Journal, we present with health and those that, under the four articles examining different right conditions, may cause disease. aspects of the oral microbiome.

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Dental Calculus and the Evolution of associated with health. In the article Uncultured Members of the Oral the Oral Microbiome on page 421, Patricia Diaz DDS, PhD, Microbiome How has the human oral and coauthors Anilei Hoare, PhD, Until 16S rRNA molecular methods microbiome changed over the past and Bo-Young Hong, PhD, describe were developed for identifi cation of 7,000 years? Did the change from shifts in the subgingival microbiome all bacteria, we knew only about those hunter-gather to farmer affect our in periodontal diseases. Transitions that could be cultivated. In the 1990s oral microbiome? Have organisms from health to disease and from molecular methods revealed that more like Porphyromonas gingivalis and gingivitis to periodontitis involve than half of the 700 bacterial species Streptococcus mutans been part of our microbial successions and adaptations in the oral cavity had not yet been oral microbiomes for millennia or to changing conditions and host cultivated. Over the past 15 years, have they recently been acquired? If responses in the gingival sulcus. through the concerted efforts of many only we could go back hundreds or oral microbiologists, 68 percent of oral thousands of years and obtain oral Understanding Caries From the Oral bacterial species in the mouth have plaque samples, we could answer these, Microbiome Perspective been cultured. This contrasts with far and many other, questions. It turns out Caries is a disease strongly lower percentages of bacteria cultured that there are time capsules available infl uenced by intake of dietary sugars from other body sites such as the gut to us that can take us back thousands and carbohydrates. Despite advice from and skin. When a bacterium is cultured, of years — the dental calculus on the dentists and public health offi cials, it can be fully characterized, used teeth of ancient skulls and mandibles. many Americans and their children in in vivo and in vitro experiments, Fortuitously, as calculus forms, it traps continue to ingest excessive amounts of manipulated by knocking out or human and microbial DNA. In the sugars and develop widespread carries. replacing genes and formally named. In article on the next page, Christina Fluoridated water and application the article on page 447, William Wade, Warinner, PhD, describes her studies of of topical fl uoride has signifi cantly BSc, PhD; Hayley Thompson, BSc, ancient calculus and provides insight reduced caries in the majority of the PhD; Alexandra Rybalka, BSc, PhD; into human health and disease and our population, but caries still remains and Sonia Vartoukian BDS, FDS, PhD, evolving microbiome from prehistoric high in disadvantaged populations. describe efforts to culture all members times to the present day. The fi elds of The article on page 437 by Anne of the oral microbiome, explain why anthropology and molecular biology Tanner, BDS, PhD; Christine Kressirer, some bacteria are very diffi cult to have joined and are giving us a PhD; and Lina Faller, PhD, reviews culture and describe which groups much better understanding of human the microbiota in caries and how our of bacteria remain uncultivated. ■ evolution, biology and human history. understanding of the caries process has evolved to include the roles that Subgingival Microbiome Shifts and benefi cial bacteria play in mitigating Community Dynamics in Periodontal acid produced in response to sugar Diseases exposure. Caries is now recognized Our understanding of periodontal as involving not only Streptococcus disease has changed markedly in the mutans, but also many acidogenic and past 50 years. We have gone from aciduric species and the concomitant being able to identify only about loss of many benefi cial species. 100 oral bacteria using cultivation Microbiome studies using molecular methods to being able to precisely and tools now allow investigators to rapidly identify the more than 700 follow microbial shifts and analyze bacterial species that comprise the changes in metabolism with high oral microbiome. We have not only precision. This greater understanding identifi ed key pathogens, but have may lead to therapies that shape come to recognize the importance the composition of the microbiome of commensals and those species and ultimately reduce caries.

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Dental Calculus and the Evolution of the Human Oral Microbiome

Christina Warinner, PhD

ABSTRACT Characterizing the evolution of the oral microbiome is a challenging, but increasingly feasible, task. Recently, dental calculus has been shown to preserve ancient biomolecules from the oral microbiota, host tissues and diet for tens of thousands of years. As such, it provides a unique window into the ancestral oral microbiome. This article reviews recent advancements in ancient dental calculus research and emerging insights into the evolution and ecology of the human oral microbiome.

AUTHOR

Christina Warinner, PhD, is he human oral cavity is home to a major public funding initiatives, such as co-director of the Laboratories diverse ecology of microorganisms, the National Institutes of Health’s Human for Molecular Anthropology collectively known as the oral Microbiome Project (HMP).6 However, and Microbiome Research (LMAMR) and a presidential microbiome. Composed of the fact that the oral microbiome will 1 research professor at the more than six hundred taxa, cause dental disease in a majority of University of Oklahoma, Tthe oral microbiota plays a central role individuals during their lifetimes has been where she holds an assistant in dental health, and increasingly it has argued to suggest that even the “healthy” professor appointment in the been shown to infl uence extraoral organs or “normal” oral microbiome today is department of anthropology. 2 7-9 She is also a guest associate and tissues, as well as general health. already in an altered dysbiotic state, 10-12 professor in the Center Investigating the evolution of this rich and likely has been for some time. for Biological Sequence microbial ecology is challenging, but it To better understand the oral Analysis at the Technical is critical for understanding the origins microbiome and its associations to University of Denmark and and prevalence of oral and systemic both dental disease and other so-called a research affi liate of the Institute of Evolutionary disorders as diverse as dental caries, “diseases of civilization,” it is useful Medicine at the University of periodontal disease, rheumatoid arthritis, to investigate how our vulnerability Zurich. Dr. Warinner earned cardiovascular disease, respiratory illness to disease is related to human her master’s and doctoral and a range of infectious diseases. evolutionary history. Evolutionary degrees in anthropology at Research on what constitutes a medicine, a fi eld that integrates both Harvard University. Confl ict of Interest: None healthy or normal oral microbiome has medicine and evolutionary biology, reported. expanded dramatically over the past provides a framework for rethinking decade,3 in large part due to the advent of conventional models of oral health and high-throughput DNA sequencing4,5 and disease,13 and biological anthropology,

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a fi eld that encompasses primatology, paleoanthropology, bioarchaeology and human biology, provides a context in which to characterize the ancestral state of the human FIGURE 1A. FIGURE 1B. microbiome.14 Together, these fi elds provide pathways to better understanding the oral microbiome, and ultimately to restoring and better maintaining oral health in the future. One of the most exciting recent discoveries in biological anthropology has come from an unlikely quarter — dental calculus (FIGURE 1).

Since at least the 1980s, it has been FIGURE 1C. FIGURE 1D. known that archaeological dental calculus contains preserved cellular structures of oral bacteria,15-17 but it was only recently discovered that it is also a robust and long-term reservoir of well-preserved DNA and proteins.17-20 Advances in ancient DNA and paleoprotein technologies now allow detailed characterization of these ancient biomolecules, enabling direct comparisons between FIGURE 1E. FIGURE 1F. ancient and modern oral microbial FIGURES 1. Archaeological dental calculus. (A,B) St. Petri cemetery, Dalheim, Germany, ca. 1100 CE. (C,D) 18,20 communities. Host proteins Sky cave tomb, Samdzong, Nepal, ca. 500 CE. (E,F) Mortuary cave, Camino del Molino, Spain, ca. 2500 BCE. preserved within ancient dental The amount of dental calculus typically required for analysis is shown in (F). Dental calculus from all three sites has calculus additionally provide insights yielded genetic data suitable for ancient microbiome analysis.20,77 into past microbial virulence and host immune response,20 and food particles entrapped within dental Dental Calculus and Ancient uncommon to observe heavy calculus calculus offer unprecedented insights Biomolecules deposits in excess of 100 mg, especially into the diets of past populations.20-23 Dental calculus is a complex, mineralized from post-Neolithic periods (FIGURE 1). This article provides an overview bacterial biofi lm that forms from dental Importantly for archaeology, dental calculus of recent ancient dental calculus plaque on the surfaces of teeth.24,25 It is found preserves over time at least as well as bone research and discusses four areas in in all known past and present human and dentine, and it has even been found on which the investigation of ancient populations, and it is nearly ubiquitous the teeth of Neanderthals28 and australo- oral microbiota is poised to make among adults without active dental pithocenes,29 in addition to humans. Among signifi cant contributions to an hygiene.17 The amount of dental calculus primates, the oldest known calculus to date evolutionary perspective on human buildup on the dentition varies widely was reported on the dentition of a Miocene health and disease: dental caries; among populations26,27 and may result from a orangutan ancestor and dates to roughly periodontal disease, rheumatoid complex combination of factors related to twelve million years ago30 (FIGURE 2). arthritis and cardiovascular disease; subsistence, dietary abrasiveness, dental Dental calculus is thus noteworthy for its respiratory infections and meningitis; hygiene practices and genetic predisposition. availability in the fossil record throughout and major infectious diseases. However, prior to modern dentistry it is not the entirety of human evolution.

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12 2 6000 5000 4000 3000 2000 1000 0 1000 2000 MYA MYA BCE BCE BCE BCE BCE BCE CE CE

Period Paleolithic Neolithic Iron Age Early Modern Mesolithic Bronze Age Medieval Industrial Earliest reported calculus Chalcolithic in primate lineage Earliest plant microfossils recovered from calculus Calculus analyzed to date using protein mass spectrometry Calculus analyzed to date using targeted DNA sequencing Calculus analyzed to date using metagenomic DNA sequencing Earliest detection of S. mutans to date Earliest detection of P. gingivalis to date First genome reconstruction of an ancient periopathogen Calculus shown in Figure 1

Bacteria shown in Figure 3

FIGURE 2. Idealized chronology of European prehistory and timeline of major ancient dental calculus fi ndings to date. Includes data from Europe,18,20,23,77 Asia,23,30,77 Africa,23,29,77 North America34,77 and South America.19 Note that regional European chronologies diff er, with major periods typically beginning earlier in southeastern Europe and later in northwestern Europe. African, Asian and New World chronologies are not shown.

Dental calculus formation occurs analyses of archaeological dental calculus lasting mark in the macroarcheological when dental plaque undergoes periodic have found that it typically contains ten- record provide opportunities to address mineralization events, although the timing to thousandfold more DNA than bone or questions that were previously thought to and triggers for this process are not well dentine from the same individual, making be unanswerable. By using these new and understood.17 During mineralization, it the richest source of ancient DNA yet emerging techniques, dental calculus can calcium phosphate ions from saliva identifi ed in the archaeological record.17 be used to address fundamental questions and gingival crevicular fl uid precipitate A number of recent studies have about the evolution of human oral health. within the dental plaque matrix, at once explored the potential of dental calculus killing the microbiota and calcifying the to reconstruct aspects of ancient health Oral Microbiota and Disease — microbial cells and other debris in situ and disease (FIGURE 2) and have shown Targets for Discovery (FIGURE 3A). Organic structures preserve it to be an area of great potential. Made well in this environment (FIGURE 3B), and possible by dramatic advances in ancient Dental Caries bacterial cell walls with functional cell DNA32 and paleoprotein33 technologies Dental diseases, such as caries and surface protein epitopes, as well as delicate over the past decade (FIGURE 4), these periodontal disease, are among the most dietary microfossils such as starch granules, studies have uncovered specifi c aspects of prevalent diseases affecting industrialized have been found to remain intact for individuals’ health states,18-20 diets,20,23,28 societies.7 In the 1960s, the average more than 10,000 years.28,31 Genetic ancestry34 and even craft activities35 using number of decayed, missing and fi lled teeth material also survives within dental a combination of genetic, proteomic and (DMFT index) among 12-year-old children calculus (FIGURE 3C), in part because the microscopic analyses of dental calculus. in Western Europe was greater than 5, and hydroxyapatite mineral within dental This type of specifi city and the ability by age 15 the average DMFT exceeded calculus strongly binds DNA.17 Genetic to address issues that typically leave no 10.36 Thus, a European child reaching

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FIGURE 3A. FIGURE 3B. FIGURE 3C.

FIGURES 3. Micrographs of ancient bacteria embedded within archaeological dental calculus dating to the medieval period, ca. 1100 CE.20 (A) Numerous calcifi ed bacterial cells are visible within dental calculus using scanning electron microscopy (SEM) with backscattered electron imaging. (B) Staining of decalcifi ed dental calculus thin sections reveals the presence of both Gram-positive and Gram-negative bacterial cells with relatively intact cell walls. (C) Hoechst stain, a blue fl uorescent dye that binds double-stranded DNA, reveals the presence of abundant genetic material. adulthood in the mid-20th century could Numerous studies have documented to continuously engage in such behavior expect one-third of his or her dentition increases in caries frequency with the has been argued to be a sign that even to be compromised by dental decay. onset of agriculture.12,41 The Eastern the “healthy” oral microbiome today From the 19th century to the mid-20th Woodlands of North America is perhaps is in an altered state of dysbiosis.7,11 century, dental decay was perceived as so the most intensively studied region, One question that emerges with these inevitable that complete dental extraction and analysis of caries frequency at observations is to what degree the oral and replacement with dentures became a 180 sites reveals that during the 2,000 microbiota of these populations changed popular gift for young women and brides years preceding the introduction of in step with subsistence practices. Recent in Western Europe and parts of North maize agriculture the percentage of research on the gut microbiome has America.37-39 Extensive dental public teeth affected by dental caries was identifi ed major changes in microbial health interventions and the introduction approximately 2 to 5 percent. After ecology associated with foraging, of fl uoridated dentifrices over the past the introduction of maize ca. 500 CE, agricultural and industrial lifestyles,52 but 50 years have signifi cantly reduced caries caries frequencies increased steadily from less is known about the oral microbiome rates,36 but even today dental caries affect 14 percent during the Late Woodland of traditional societies.53,54 A recent study more than 40 percent of children and 90 period to 18 percent during the late of ancient oral microbial communities percent of adults in the United States.40 Prehistoric period to 22 percent during spanning the past 8,000 years reported Dental caries are easily observed in the early Contact period12 (FIGURE 5). evidence for slight, phylum-level microbial the archaeological record, and they have Although not all regions saw such sharp shifts correlated with the onset of been systematically studied for more than increases, a general trend toward increased agriculture and industrialization;18 deeper a century.41 Extensive data have now caries frequency in agricultural populations sequencing and a larger sample size in been collected on paleoanthropological is observed globally,12,41 with relatively future studies is anticipated to clarify these and archaeological populations around few exceptions.47-50 associations and provide greater taxonomic the world, spanning time periods from The origin of today’s extreme levels resolution during these transitions. the Pleistocene42 to the 19th century.43 of dental decay, however, lies not with Direct investigation of ancient It is clear from these studies that diet is agriculture, but with the introduction of carious lesions using ancient DNA the major driver of caries frequency.12 refi ned fl ours and sugars during the Early techniques has identifi ed the presence of Although caries are observed during Modern period (ca. 1450-1800 CE; FIGURE the cariopathogen Streptococcus mutans,55 all time periods, and are, in fact, also 2). In Western societies, tooth decay was but reconstructing the full polymicrobial present in nonhuman primates,44,45 caries so severe during this time that smiling is all community contributing to such lesions56 frequencies vary remarkably through but absent from European and American is challenging because dentine is highly time and space. The most salient patterns portraiture of the period.51 Since then, susceptible to postmortem alteration by relate to the transition between foraging consumption of refi ned carbohydrates environmental microbes.20 By contrast, (hunting and gathering) and agriculture,46 has been met with increasingly aggressive dental calculus is much better preserved.20 and later to the widespread availability oral hygiene regiments and prophylactic Genetic sequences from S. mutans have of refi ned fl our and sugar, especially dental care as necessary measures to been detected in the dental calculus of sucrose, during the 18th century.41 prevent premature tooth loss.24 The need diverse populations,18-20 but interestingly

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25% ■ Late Archaic ■ Early Woodland

20% ■ Middle Woodland ■ Late Woodland ■ Late Prehistoric 15% ■ Post-Contact

10% Caries frequency

FIGURE 4. Extraction of genetic material from archaeological dental calculus requires highly 5% specialized ancient DNA laboratories to prevent contamination from modern sources. 0% 1500 1000 500 0 500 1000 1500 2000 it has not been detected in samples prior BCE BCE BCE CE CE CE CE to the Bronze Age (ca. 2200-1000 BCE)18 (FIGURE 2). Recent phylogenetic analysis of modern S. mutans strains has estimated that S. mutans underwent an exponential FIGURE 5. Dental caries frequency in prehistoric Eastern Woodland populations of North America. A dramatic expansion ca. 10,000 years ago (95 percent increase in caries frequency is associated with the adoption of maize agriculture ca. 500 CE.12 confi dence interval: 3,268–14,344 years ago), suggesting an association with the onset of agriculture in the Near East.57 with periodontitis,66 has recently been the descriptions are usually qualitative Future ancient DNA investigations of implicated in the development and and limited to small case reports.12 At dental calculus using whole-genome progression of rheumatoid arthritis,67,68 present there have been no large-scale, capture enrichment technologies32 may and treatment of periodontitis has been systematic studies of periodontal disease be able to reveal the natural history found to alleviate rheumatoid arthritis prevalence in the past, leaving many gaps of S. mutans and determine the major symptoms.69,70 Periodontitis is also in our understanding of the antiquity, drivers of its evolution and functional associated with a 19 percent increase in prevalence and signifi cance of periodontal role within the oral cavity of humans. the risk of cardiovascular disease,71 and a disease in human evolutionary history. recent study of cardiovascular specimens Although clinical metrics73 are diffi cult Periodontal Disease, Rheumatoid Arthritis found that >80 percent of diseased heart to apply to archaeological specimens, and Cardiovascular Disease valves and >90 percent of aortic aneurisms molecular characterization of periodontal Periodontal diseases affect up to 90 have been infected with cariogenic or disease is increasingly feasible. Recent percent of the worldwide population,9 and pathogenic periodontal bacteria.62 conceptual changes in periodontology over moderate-to-severe chronic periodontitis Although there is ample evidence the past 50 years now point to disrupted is estimated to affect 13 percent of U.S. for periodontitis in the archaeological microbial communities and host-mediated adults older than age 30,58 although new record, periodontal diseases are very poorly infl ammatory tissue destruction as the diagnostic criteria suggest that this may studied. The most widely used laboratory proximate causes of periodontal disease,74,75 be a gross underestimate.59 In addition guide for osteological analysis, Standards and new molecular approaches, such to dental morbidities, periodontitis for Data Collection From Human Skeletal as metagenomics and metaproteomics, is also associated with increased risk Remains,72 contains recording metrics for allow culture-free investigation and of a wide range of so-called “diseases age estimation from dental development comparison of oral microbiota and host of civilization,” including type II and wear, enamel hypoplasia measurement, immune response from both clinical and diabetes, obesity, rheumatoid arthritis, caries scoring and dental calculus archaeological dental samples (FIGURE 2). cardiovascular disease, stroke and quantifi cation, but no information on how For example, a recent study of medieval pulmonary disease.9,60-65 This association to record or measure periodontal disease dental calculus found that periopathogens may refl ect a partially causal relationship. in archaeological dentitions. As a result, common today were also associated with For example, Porphyromonas gingivalis, it is rarely noted in archaeological reports. suspected periodontal disease cases nearly an oral bacterium strongly associated When periodontal disease is mentioned, a thousand years ago.20 Frequencies of

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the periodontal pathogens P. gingivalis, rheumatologist Bruce Rothschild, MD, cultures, from the elites of ancient empires Tannerella forsythia, Treponema denticola proposed a radical hypothesis — that to prehistoric hunter-gatherers. Calcifi c and Filifactor alocis were found to be highly rheumatoid arthritis is a vector-transmitted atherosclerosis has been identifi ed, fi rst by elevated in dental calculus collected from infectious disease that originated in autopsy and later by X-ray and computed archaeological dentitions with generalized central North America and spread to tomography (CT), in the coronary and moderate or severe attachment loss. the Old World after European contact peripheral arteries of mummies originating Additionally, virulence factors expressed in the region ca. 1750.78,79 Among the from ancient cultures in Egypt, the by these taxa and host innate immune evidence he assembled, he noted that Alps, Peru, the American Southwest, proteins were found at high abundance the fi rst documented case of rheumatoid the Aleutian Islands and Korea.83,84 among the proteins recovered from these arthritis in Europe dates to 1800, while Moreover, radiological scans suggest samples, strongly suggesting periodontitis- cases in North America are known that the prevalence of atherosclerosis associated infl ammation. In dental calculus from archaeological remains up to 6,500 was relatively high in prehistory, and, from one individual, genetic sequences years old, and that Native Americans in the case of Egypt, no signifi cant for T. forsythia were so abundant that a today have unusually high rates of the difference was found in the incidence near complete ancient genome for this or prevalence of atherosclerotic plaques pathogen could be reconstructed. The between today and 3,500 years ago.85 ancient strain was found to harbor the A collaboration of cardiologists, same 14 virulence proteins found in Cardiovascular disease is radiologists, molecular biologists and modern T. forsythia, but it lacked several conventionally viewed as archaeologists known as the Horus mobile elements, including a putative a disease of modernity, study team has been at the forefront of antibiotic resistance gene, tetQ, found ancient atherosclerosis research since today in the T. forsythia reference strain. but archaeological evidence 2008.83 Because many of the risk factors Two additional studies have begun to now confi rms its presence in associated with cardiovascular disease look at temporal changes in periodontal diverse ancient cultures. today do not apply to ancient populations, pathogens. In a study of ancient Chilean they have proposed that the high rates of and Argentinian dental calculus, P. gingivalis atherosclerosis in the past may have resulted was detected in most time periods from from chronic systemic infl ammation caused 2500 BCE to the present,19 and in a study autoimmune disease (more than fi vefold by long-term infection by gastrointestinal of ancient Europeans, P. gingivalis was higher than the U.S. general population80). parasites or repeated exposure to microbial detected in all major periods dating back Although aspects of this hypothesis are or viral pathogens;83,86 however, in the to the Mesolithic, ca. 5550 BCE-3450 now quite dated,81 the recent link made absence of direct evidence for such BCE18 (FIGURE 2). Interestingly, the latter between rheumatoid arthritis and P. infections, they note that other yet-to-be study found that P. gingivalis frequencies gingivalis67,68 has rekindled interest in a discovered risk factors may also play a role. were lower in Mesolithic hunter-gatherers possible microbial origin for this disease. Poor oral health may be just such a risk than in post-Neolithic farmers, a fi nding By using ancient DNA from dental factor. Untreated caries, heavy calculus consistent with observations that hunter- calculus along with osteological data, it deposits and alveolar recession suggest both gatherer societies in the recent past may be possible to unravel the historic dysbiotic oral microbial communities and and today appear to have lower rates of biogeography of virulent P. gingivalis strains sustained infl ammation were prevalent in periodontal disease than agricultural and and their relationship to autoimmunity. many ancient populations. A promising industrialized societies.11,76 However, further Finally, the recent link made between future direction in this research would research is necessary to ensure that this cardiovascular disease and periodontal be to genetically test ancient calcifi c fi nding is not simply an artifact of more and cariogenic taxa82 raises questions as atherosclerotic plaques directly for the advanced DNA decay in older samples.77 to the antiquity of oral involvement in presence of oral taxa. Systematic CT The investigation of P. gingivalis atherosclerotic plaques. Cardiovascular scanning has identifi ed atherosclerotic in archaeological dental calculus may disease is conventionally viewed as a disease plaques in more than 50 ancient mummies also have implications for the study of modernity, but archaeological evidence from around the world, and biopsies of rheumatoid arthritis. In 1990, now confi rms its presence in diverse ancient of these plaques could be analyzed and

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compared to microbial profi les generated in aspiration pneumonia,95,96 the leading and/or other respiratory infections were from dental calculus collected from the cause of death and the second most a major cause of human mortality in same individuals. Taxonomic matches common cause of hospitalization among prehistory.99 The discovery that dental would be strong evidence for a long- nursing home patients.96 Additionally, it is calculus is a reliable source of genetic term role of oral involvement in the common for the elderly to acquire extraoral material from common respiratory initiation of cardiovascular disease. pathobionts, such as Staphylococcus aureus, pathobionts opens up the possibility Pseudomonas aeruginosa and Escherichia of conducting epidemiological studies Respiratory Infections and Meningitis coli, in their dental plaque, which also of past carriage rates, and also presents The oral microbiome is the natural contribute to aspiration pneumonia.2 the opportunity to investigate the reservoir for a large number of pathobionts To date, multiple pathobionts have evolution of these taxa through time. (endogenous potential pathogens), been identifi ed in archaeological dental Dental calculus may also help to date including Streptococcus pneumoniae, calculus, including S. pneumoniae, H. the origin of infectious diseases that have Haemophilus infl uenzae, Moraxella infl uenzae, S. pyogenes, C. diphtheriae and recently emerged from the oral cavity, such catarrhalis, Streptococcus pyogenes, N. meningitidis.20 Additionally, genetic as bacterial meningitis and gonorrhea. Corynebacterium diphtheriae, Bordetella Phylogenetic studies of Neisseria, a genus of pertussis and Neisseria meningitidis,1 bacteria that colonize mucosal surfaces in which cause a wide range of acute and animals, indicate that N. meningitidis and potentially life-threatening respiratory Dental calculus may also N. gonorrhoeae belong to a recently diverged and meningeal infections, especially in help to date the origin of pathogenic clade in humans that is most children. Importantly, asymptomatic infectious diseases that have closely related to the common nasopharynx carriage of these microbes is relatively commensal Neisseria lactamica;103-105 prevalent, although it varies widely recently emerged from the oral however, the timing and context of this among populations. For example, reported cavity, such as bacterial divergence are not well understood. Both carriage rates of S. pneumonia range from meningitis and gonorrhea. N. meningitidis and N. gonorrhoeae are 1 percent in parts of Scandinavia to more obligate human taxa, indicating that this than 80 percent in parts of France and divergence must have occurred since the Gambia, while H. infl uenzae carriage chimp-human split approximately 6 million rates range from 3 percent in Sweden to sequences consistent with Bordetella years ago, and whole-genome comparisons 88 percent in Costa Rica, and carriage parapertussis, an organism responsible with other Neisseria species suggest that rates for M. catarrhalis, a common cause for persistent cough in children that is N. meningitidis may have undergone of middle ear infections, range from 2 similar to pertussis,97 and several species a population bottleneck and acquired percent in parts of Sweden to 82 percent of Moraxella were also identifi ed. These its virulence genes for polysaccharide in parts of the Netherlands.87 In Europe, pathobionts were detected in the dental capsule synthesis very recently, perhaps N. meningitidis is typically carried by an calculus of two individuals excavated from only a few centuries ago.105 Nonspecifi c average of 24 percent of teenagers and 8 the medieval St. Petri cemetery in Dalheim, endocranial meningeal reactions are often to 13 percent of adults,88 and nearly one- Germany (ca. 1100 CE). Although there found in skeletons,106 and with the recent fi fth of American schoolchildren carry S. is no evidence that these organisms were identifi cation of putative N. meningitidis pyogenes at any given time.89 For pathogens causing disease in these individuals, genetic sequences in archaeological dental with widespread vaccine programs, they nevertheless provide the fi rst direct calculus, this hypothesis has become such as Corynebacterium diphtheriae and evidence of respiratory pathobiont carriage testable. Future ancient DNA investigations Bordetella pertussis, carriage rates are in the oral cavity before the 20th century.20 using whole-genome capture enrichment generally low,90-94 but were presumably Only a century ago, respiratory technologies32 hold great promise for once much higher. In addition to these infections were the leading causes of death resolving the origins and evolution of pathobionts, even commensal oral taxa in the U.S.,98 and studies of periosteal N. meningitidis, a pathobiont whose pose serious health risks among the elderly rib lesions in skeletal collections99 and mortality rate from bacterial meningitis and immunocompromised. Aspiration of soft tissue changes in South American and septicemia continues to exceed 10 common oropharyngeal taxa can result mummies100-102 suggest that pneumonia percent even in developed nations.104

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Major Infectious Diseases greatly to what is known about the origins using bone samples, including treponemal Finally, although not true members of of a handful of pathogens, including M. diseases such as venereal syphilis,125 which is the oral microbiome, several opportunistic tuberculosis,115,116 Yersinia pestis,117-119 and H. of particular importance given its historical and obligate pathogens can be found pylori,120 and it has been used to confi rm the and clinical signifi cance,125 as well as its past transiently within dental plaque, buccal presence of several additional pathogens in intractability to ancient DNA analysis.126,127 mucosa and saliva.1 These include ancient infections, including M. leprosy,121 Although none of the above the causative agents of tuberculosis T. pallidum122 and Variola virus.123 pathogens has yet been identifi ed from (Mycobacterium tuberculosis), leprosy However, genetic detection rates for archaeological dental calculus, the fact (Mycobacterium leprae), plague (Yersinia most ancient pathogens are low, even in that so many infectious agents transiently pestis), syphilis (Treponema pallidum), remains with overt and relatively diagnostic inhabit the oral cavity during disease gastritis (Helicobacter pylori) and smallpox paleopathology indicators. Antemortem progression makes future detection of (Variola virus), among others. M. tissue destruction likely enhances pathogens from dental calculus at least tuberculosis, for example, is present in postmortem decay, which may contribute plausible. If successful, such analyses sputum and regularly comes into contact to poor preservation of pathogen DNA could greatly expand our understanding with the oral cavity throughout the entire of human pathogen evolution. course of the disease.107 In addition to sputum, M. tuberculosis has also been Conclusion detected in 92 percent of dental plaque Because dental calculus calcifi es The incorporation of genetic samples from infected patients using during life, it does not undergo material from commensal, pathobiont polymerase chain reaction (PCR)-based the same decomposition and pathogenic microorganisms into techniques.108 Leprosy involves the oral dental plaque, and later dental calculus, cavity in up to 60 percent of cases, and processes as the rest of the presents a rare opportunity to study the multiple oral structures may develop body, and it is nearly ubiquitous evolution of the human oral microbiome lesions and ulcers, including the hard and in skeletal collections. and associated diseases in archaeological soft palate, the gingiva, tongue, lips and skeletal collections spanning thousands buccal mucosa.109,110 During outbreaks of years. Great progress has already of bubonic plague, Y. pestis that escapes been made in developing the tools and the lymphatic system and infects the within infected bone, and examples of technologies necessary to extract genomic lungs causes pneumonic plague, a highly well-preserved soft tissue are relatively rare and proteomic information from ancient infectious form of the disease that results in outside of a few geographic regions. Dental dental calculus, and clinical research on the lethal fulminant pneumonia.111 T. pallidum calculus presents a promising alternative for oral microbiome is laying the theoretical is known to cause oral lesions during the screening ancient skeletons for infectious foundations for making this information secondary phase of syphilis infection, pathogens. Because dental calculus calcifi es relevant in today’s dental practices which may last for many years,112 and this during life, it does not undergo the same and hospitals. Through collaborations provides ample opportunity for passive or decomposition processes as the rest of between oral health science and ancient active incorporation and preservation in the body, and it is nearly ubiquitous in dental calculus research, we can leverage calcifying dental plaque biofi lms. Finally, skeletal collections.124 DNA within dental knowledge of the ancestral oral microbiome H. pylori is readily found in the saliva and calculus has been shown to preserve well to improve human health today. ■ dental plaque of infected individuals,113 and over long time scales, and because it forms 114 ACKNOWLEDGMENTS smallpox causes oropharyngeal lesions. incrementally, serially entrapping microbes This work was supported by grants from the National Science Infectious diseases have played a major and debris from discrete periods of time,17 it Foundation (BCS-1516633 and BCS-1523264 to C.W.). 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Subgingival Microbiome Shifts and Community Dynamics in Periodontal Diseases

Patricia I. Diaz, DDS, PhD; Anilei Hoare, PhD; and Bo-Young Hong, PhD

ABSTRACT High-throughput 16S rRNA gene sequencing has allowed the characterization of subgingival microbiome shifts from health to periodontitis identifying health-associated, periodontitis-associated and core species, which preserve their proportions from health to disease. The development of gingivitis is also characterized by distinct shifts. Microbiome shifts resemble microbial successions and result from interspecies interactions and community adaptation to the changing environment as infl ammation ensues. Gingivitis-associated and core species are proposed as likely mediators of microbiome transitions.

AUTHORS

Patricia I. Diaz, DDS, Anilei Hoare, PhD, is a eriodontitis is one of the most host and it is, therefore, the interplay PhD, is an associate postdoctoral fellow in the prevalent chronic conditions in between the subgingival community professor in the division division of periodontology, humans with 46 percent of U.S. of microorganisms and local immune of periodontology, department of oral health adults suffering from the disease responses that ultimately drives bone department of oral health and diagnostic sciences 4 and diagnostic sciences at UConn Health in and about 9 percent presenting and connective tissue attachment loss. at UConn Health in Farmington, Conn. Pwith a severe form.1 Because the oral The subgingival microbiome, that is, Farmington, Conn. Confl ict of Interest cavity is easily accessed, a large body the community of microorganisms that Confl ict of Interest Disclosure: None reported. of work exists trying to uncover the inhabits the subgingival environment, Disclosure: None reported. etiopathology of this condition. Several has been the subject of investigation for Bo-Young Hong, PhD, is a postdoctoral fellow in the lines of evidence indicate that bacteria many decades. Research comparing the division of periodontology, are necessary for the development of subgingival microbiota under different department of oral health infl ammation in the periodontal tissues. periodontal conditions has been conducted and diagnostic sciences This evidence includes the classic using various techniques. Most notable at UConn Health in experimental gingivitis studies in humans historic studies prior to the advent of 16S Farmington, Conn. Confl ict of Interest by Löe et al., evidence of the resolution ribosomal RNA (rRNA) gene sequencing Disclosure: None reported.. of infl ammation after periodontal are summarized at the top of TABLE 1. These treatment involving mechanical studies were of exceptional quality and were debridement and animal models showing performed with what, at the time, were lower levels of bone loss in germ-free state-of-the-art methods to characterize the and antibiotic-treated animals.2,3 Tissue oral microbiota. The techniques available destruction, however, is mediated by the at the time, however, did not allow a global

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TABLE 1

Classic Studies of the Subgingival Microbiome in Different Periodontal Conditions Prior to the High-Throughput Sequencing Era

Reference Technique Key fi ndings

Listgarten, 19765 Compared plaque from diff erent ■ Diff erences in plaque thickness and composition between health, gingivitis and periodontal conditions using light chronic periodontitis. and electron microscopy. ■ Health: Gram-positive cocci predominate. ■ Gingivitis: Coccoid, fi lamentous, fl agellated and spirochaete forms found; mixture of Gram- positive and Gram-negative. ■ Periodontitis: A distinct bacterial population with Gram-negative, fl agellated and presumably motile forms, rich in spirochetes and bristle brush formations.

Armitage et al., 19826 Compared plaque from diff erent ■ Plaque load of spirochaetes positively correlated with periodontal disease severity. periodontal conditions using dark fi eld microscopy.

Moore and Moore, Compared plaque from diff erent ■ Five hundred and nine taxa identifi ed, 368 of which were detected more than once. 19948 periodontal conditions via ■ Complexity of subgingival communities increases from health to disease. cultivation. ■ Actinomyces spp., Veillonella parvula, Streptococcus spp., Capnocytophaga gingivalis, Eubacterium saburreum, Aggregatibacter spp., Leptotrichia D16, Neisseria spp. and Rothia dentocariosa were increased in health. ■ Fusobacterium nucleatum, Filifactor alocis, Eubacterium spp., Campylobacter spp., Dialister pneumosintes, Prevotella spp., Peptostreptococcus spp., Porphyromonas gingivalis, spirochaetes and a Mycoplasma sp. increased with disease severity (health to gingivitis to periodontitis).

Socransky et al., 19987 Evaluated 40 species in subjects ■ Identifi cation of fi ve complexes of tightly associated species. with and without periodontitis ■ The red complex (Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola) via checkerboard DNA-DNA showed a strong positive correlation with periodontitis severity. hybridization. Studies based on 16S rRNA gene sequencing via Sanger methods

Kroes et al., 199947 Evaluated the subgingival ■ First study to demonstrate the utility of 16S rRNA gene sequencing in subgingival microbiota microbiome via 16S rRNA gene characterization. cloning and Sanger sequencing ■ 77 species/phylotypes identifi ed, 48 percent of which were novel. and via cultivation in one subject ■ More novel phylotypes identifi ed via 16S rRNA gene sequencing than by cultivation. with gingivitis.

Paster et al., 200111 Evaluated the subgingival ■ First study to provide a detailed molecular characterization of the subgingival microbiome in microbiome via 16S rRNA gene several subjects. cloning and Sanger sequencing ■ A total of 347 species/phylotypes identifi ed, 215 of which were novel, with the number of in subjects with diff erent species in subgingival plaque estimated to be ~415. periodontal conditions.

Kumar et al., 200512 Evaluated the subgingival ■ A total of 274 species/phylotypes identifi ed. microbiome via 16S rRNA gene ■ First controlled study comparing health and chronic periodontitis via 16S rRNA gene cloning and Sanger sequencing sequencing. in health and periodontitis. ■ Identifi ed species/phylotypes associated with periodontitis: Peptostreptococcus spp., Filifactor alocis, Megasphaera sp., Desulfobulbus sp., Dialister spp. Campylobacter spp., Selenomonas sp., Deferribacteres sp., Catonella sp., Tannerella forsythia, Streptococcus spp., Atopobium sp., Eubacterium sp. and Treponema sp. ■ Identifi ed species/phylotypes associated with health: Veillonella sp., Campylobacter gracilis, Campylobacter showae, Abiotrophia adiacens, Eubacterium saburreum, Gemella sp., Streptococcus sanguis, Streptococcus mutans, Capnocytophaga gingivalis, Rothia dentocariosa, Eubacterium sp. and Selenomonas sp.

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view of the microbial composition of a given sample together with high-resolution taxonomy and high-throughput sample processing. Microscopic techniques such as those used by Listgarten et al.5 and Armitage et al.6 did not permit taxonomic classification of species. The DNA-DNA checkerboard study by Socransky et al.7 was performed with a technique that allowed high-throughput processing of samples and, therefore, the study includes many subjects. However, the microbiota evaluation was limited to 40 species. The comprehensive cultivation study of Moore and Moore8 was limited by laborious laboratory procedures and lack of culturability of a great proportion of the subgingival microflora. Despite the discussed limitations, these studies revealed a great proportion of the subgingival microbial diversity and community dynamics and were crucial to our current understanding of periodontitis etiopathology. All of these investigations agree that periodontal disease is associated with shifts in the composition of the community of microorganisms at the subgingival crevices in comparison to health.

16S rRNA Gene Sequencing: A New Era in Periodontal Microbiology The field of microbiology took a turn after the advent of 16S rRNA gene sequencing as the method of choice to infer phylogenetic relatedness among FIGURE 1. Steps required for the characterization of the subgingival plaque microbiome via 16S rRNA gene bacterial species.9,10 rRNAs are essential sequencing. Species are represented by different colors. Conserved regions in the 16S rRNA gene are depicted in gray and are used for design of universal primers. Hypervariable regions (V1 to V9) are depicted in black and are components of the protein synthesizing used for identification of species. Arrow labeled as “a” shows steps required prior to the advent of high-throughput machinery in all cells and, therefore, sequencing. The different 16S rRNA genes from all species in the sample were amplified via PCR with universal their gene sequences have highly primers. Individual 16S rRNA gene amplicons needed to be subsequently cloned and multiplied in a host such as conserved (similar) regions across Escherichia coli to obtain a separate preparation of each cloned 16S rRNA gene. Cloned 16S rRNA genes were species (FIGURE 1). These conserved then sequenced via Sanger methods and the sequences compared to a reference database for their identification. regions are utilized to design “universal” Arrow labeled as “b” shows how high-throughput sequencing has simplified the process by allowing direct sequencing PCR primers capable of recognizing of PCR amplicons. Up until now, the most widely available sequencing technologies did not allow sequencing of the full-length 16S rRNA gene and, therefore, our group targets the first 350 base pairs containing the hypervariable segments of the 16S rRNA gene regions V1 and V2, which perform very well discriminating oral species from each other. Universal primers flanking this sequence of most, if not all, bacterial region are designed as fusion primers incorporating also a spacer (orange), an identifier (blue) and an adapter (dark species. Apart from conserved regions, gray) sequence.47 All 16S rRNA gene molecules amplified in the PCR reaction are then simultaneously sequenced. there are also several hypervariable Moreover, the addition of identifier sequence tags allows sequencing of many samples simultaneously.

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TABLE 2

Studies of the Subgingival Microbiome in Different Periodontal Conditions Using High-Throughput Sequencing of the 16S rRNA Gene

Reference Methods Key fi ndings Health versus periodontitis

Griff en et al., 201214 29 periodontally healthy controls and 29 ■ 16 phyla, 106 genera and 596 species identifi ed. 81 percent of sequences could subjects with chronic periodontitis (shallow be mapped to cultivated species. and deep sites were sampled). ■ Community profi les were diff erent in health and disease with higher diversity in periodontitis. ■ 123 species-level phylotypes were enriched in periodontitis, while 53 were enriched in health. ■ Shallow pockets of periodontitis subjects showed preponderance of disease- associated organisms. ■ The major health-associated species are suppressed but not lost in periodontitis.

Abusleme et al., 201315 22 subjects with chronic periodontitis and ■ Communities in health and periodontitis diff ered with higher diversity in periodontitis. 10 healthy controls. Periodontitis subjects ■ 46 species-level phylotypes were enriched in periodontitis and 14 were enriched sampled at two sites with 5 mm probing in health. depth (one with bleeding). Total load and ■ Defi ned core subgingival species as those present in a majority of subjects and at load of specifi c genera measured via real equal relative abundance in health and disease. F. nucleatum is the most abundant time qPCR. core species. ■ Bleeding was associated with higher bacterial load. ■ Shifts from health to periodontitis resemble ecological succession without replacement of health-associated species.

Hong et al., 201517 34 subjects with chronic periodontitis ■ Communities in health and periodontitis diff ered. compared to 79 healthy subjects sequenced ■ No demographic or medical characteristics of periodontitis subjects were associated by the Human Microbiome Project. Diabetics with specifi c microbial profi les. and subjects with chronic kidney disease ■ Two types of microbiome profi les seen in periodontitis (clusters A and B), with the included in the periodontitis group. cluster B community showing a positive correlation with periodontitis extent. ■ Two types of microbiome profi les seen in health (clusters L and S).

Kirst et al., 201516 25 subjects with chronic periodontitis and ■ Communities in health and periodontitis diff ered. 25 healthy controls. ■ 18 species-level phylotypes enriched in periodontitis and fi ve enriched in health. ■ Microbial diversity was not signifi cantly diff erent between health and periodontitis. Periodontitis versus gingivitis versus health

Park et al., 201537 12 healthy subjects, 10 subjects with ■ Distinct communities in health, gingivitis and periodontitis. gingivitis and 10 subjects with periodontitis. Health versus gingivitis

Kistler et al., 201335 20 healthy subjects abstained from oral ■ Increased community diversity and signifi cant shifts in microbiome structure after two hygiene. Sampled at baseline and after one weeks of oral hygiene abstention. and two weeks. ■ Identifi ed species-level phylotypes positively and negatively correlated with gingivitis (bleeding on probing).

Huang et al., 201436 50 subjects underwent controlled transitions ■ Temporal shifts in community structure seen along the progression from NG to from naturally occurring gingivitis (NG) to baseline to EG. healthy gingiva (baseline) to experimental ■ 15 genera could distinguish healthy and gingivitis samples with 74 percent accuracy. gingivitis (EG). ■ Shotgun metagenomic sequencing showed a functional shift with gingivitis-enriched functions such as fl agellar biosynthesis. ■ Two host types, I and II, with distinct susceptibility to gingivitis were identifi ed. Genera such as Selenomonas, Peptostreptococcus and Veillonellaceae, among others, enriched in type-II hosts.

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regions (different across species) along to the labor-intensive cloning process, have become the method of choice for the 1500 base pairs that form the 16S these studies were still not high throughput oral microbiota characterization if not rRNA gene, which can be used as and lacked the sequencing depth to cover for the existence of curated reference signatures to discriminate one species most subgingival diversity within samples. databases such as the Human Oral from another. Thus, 16S rRNA gene Microbiome Database (HOMD),13 sequencing became the tool of choice High-Throughput Sequencing and which allows the classifi cation of most to determine the global composition the Road to Commoditization of sequences in a dataset to the species of the bacterial community in a given Microbiome Evaluation level. Thus, the process of evaluating plaque sample. However, obtaining The development of 16S rRNA subgingival communities via high- the 16S rRNA gene sequences of gene sequencing opened a new era in throughput 16S rRNA gene sequencing a bacterial community containing periodontal microbiology by allowing has become accurate, cost-effective multiple species was initially a a global view of the bacterial species and accessible to most researchers. laborious process as it involved cloning in a sample, but it was not until the of 16S rRNA gene amplicons into advent of high-throughput sequencing Subgingival Microbiome in Health plasmid vectors with their subsequent and Periodontitis as Revealed by replication in Escherichia coli to obtain High-Throughput 16S rRNA a pure preparation of each 16S rRNA Gene Sequencing amplifi ed molecule. Each amplifi ed, 16S rRNA gene sequencing TABLE 2 summarizes a series of cloned and purifi ed 16S rRNA gene would not have become the pioneer studies of the subgingival type was then sequenced by Sanger method of choice for oral microbiome conducted via high- methods and its sequence compared to throughput sequencing of 16S rRNA a database of archived 16S rRNA gene microbiota characterization gene amplicons. The studies by Griffen sequences to fi nd a species match or if not for the existence of et al.,14 Abusleme et al.,15 Kirst et al.16 the closest relative (“a” in FIGURE 1). curated reference databases. and Hong et al.17 constitute the most The pioneer studies that investigated comprehensive characterizations to date the subgingival microbiome in health and of the shifts in microbiome composition under different periodontal conditions from health to periodontitis. Although via 16S rRNA gene cloning and Sanger methods that the technique became the sampling techniques and laboratory sequencing are summarized at the bottom new gold standard. Massively parallel methods somewhat differ across of TABLE 1. These studies provided for the sequencing techniques circumvent the studies, all manuscripts report profound fi rst time a global view of the subgingival cloning step and allow direct sequencing shifts in community structure (i.e., microbiome that was independent of the of 16S rRNA gene amplicons obtained species proportions) from health to cultivation requirements of species. For from a polymicrobial sample (“b” in disease. Reassuringly, the reported instance, Paster et al.11 found 347 species- FIGURE 1). Furthermore, the addition shifts agree with pre-16S rRNA gene level phylotypes in all subgingival plaque of a sequence tag to each sample during sequencing studies showing a trend samples analyzed, 215 of which were novel. the initial PCR amplifi cation makes it toward higher levels of Gram-negative Among the novel sequences were some possible to combine different biological microorganisms in periodontitis and from phyla such as Synergistetes and TM7, samples in the same sequencing a clear association of the red complex which at the time had no cultivable oral run. It is, therefore, now feasible species (Porphyromonas gingivalis, representant. Moreover, about 60 different to simultaneously characterize the Tannerella forsythia and Treponema Treponema species were identifi ed revealing subgingival microbiome composition denticola) described by Socransky et a great variety of species from this diffi cult to of many samples, at a relatively al.7 with disease.14,15 Furthermore, cultivate genus. Using similar methodology, low cost, in a short time period and high-throughput sequencing studies Kumar et al.12 statistically compared the obtaining thousands of sequences confi rmed, as was previously seen microbiota in health and periodontitis per sample to guarantee detection of via cultivation,8 that periodontitis identifying taxa strongly associated with most species present. However, 16S is associated with an increase in either condition (TABLE 1). However, due rRNA gene sequencing would not the relative abundance of a diverse

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Abiotrophia defectiva¹ Comamonadaceae nbu379c11c1¹ Porphyromonas sp. HOT279² Health- Acidovorax sp. 98-63833¹ Comamonadaceae VE2A04¹ Propionibacterium propionicum¹ associated Acinetobacter junii¹ Corynebacterium durum² Rothia aeria³ Actinobaculum sp. HOT183¹ Corynebacterium matruchotii² (1 core) Rothia dentocariosa³ (1 perio) species Actinomyces massiliensis¹ Eikenella corrodens¹ (1 core) Schwartzia sp. HOT155¹ (1 perio) Actinomyces naeslundii³ Fusobacterium nucleatum ss polymorphum¹ (1 perio) Selenomonas noxia¹ Actinomyces odontolyticus¹ Gemella haemolysans² Selenomonas sp. HOT138¹ Actinomyces sp. HOT169² Gemella morbillorum² Streptococcus B66¹ Actinomyces sp. HOT170² Granulicatella adjacens² (1 core) Streptococcus cristatus¹ Actinomyces sp. HOT171³ Haemophilus P3D1 620¹ Streptococcus gordonii¹ Actinomyces sp. HOT175² Haemophilus parahaemolyticus¹ Streptococcus intermedius² Actinomyces sp. HOT177¹ Haemophilus parainfl uenzae¹ (1 core) Streptococcus mitis bv2¹ Actinomyces sp. HOT180¹ Kingella oralis¹ Streptococcus mitis² (1 core) Arthrobacter woluwensis¹ Lautropia AP009¹ Streptococcus sanguinis³ Bergeyella sp. HOT322¹ Lautropia mirabilis² (1 core) Streptococcus sp. HOT058¹ Brachybacterium rhamnosum¹ Leptotrichia sp. HOT212¹ Streptococcus sp. HOT064¹ Burkholderia cepacia¹ Leptotrichia sp. HOT225¹ Streptococcus vestibularis¹ Capnocytophaga gingivalis² Moraxella osloensis¹ unclassifi ed Xanthomonadaceae¹ Capnocytophaga leadbetteri¹ Neisseria elongata¹ Veillonella parvula² (1 core) Capnocytophaga sputigena¹ Porphyromonas catoniae²

Campylobacter gracilis² Fusobacterium nucleatum ss nucleatum¹ Prevotella nigrescens¹ Core Catonella morbi¹ Fusobacterium nucleatum ss vincentii² (1 perio) Prevotella sp. HOT317¹ species Corynebacterium matruchotii¹ (2 health) Granulicatella adjacens¹ (2 health) Pseudomonas pseudoalcaligenes¹ Eikenella corrodens¹ (1 health) Haemophilus parainfl uenzae¹ (1 core) Streptococcus mitis¹ (2 health) Fusobacterium nucleatum ss animalis¹ (1 perio) Lautropia mirabilis¹ (2 health) Veillonella parvula¹ (2 health)

Aggregatibacter sp. HOT458¹ Lachnospiraceae [G-8] sp. HOT500² Schwartzia sp. HOT155¹ (1 health) Periodontitis- Anaeroglobus geminatus² Leptotrichia EX103¹ Selenomonas dianae² associated Anaerolineae [G-1] sp. HOT439² Leptotrichia IK040¹ Selenomonas sp. HOT126¹ species Atopobium rimae¹ Leptotrichia sp. HOT215¹ Selenomonas sputigena³ Bacteroiaceae [G-1] sp. HOT272² Leptotrichiaceae [G-1] sp. HOT210¹ Shuttleworthia C1¹ Bacteroidales [G-2] sp. HOT274³ Mogibacterium timidum² Solobacterium moorei¹ Bacteroidetes [G-3] sp. HOT280¹ Mycoplasma faucium¹ Stomatobaculum sp. HOT373¹ Bacteroidetes [G-6] sp. HOT516¹ Parvimonas micra³ Streptococcus anginosus¹ Bifi dobacterium dentium¹ Peptostreptococcaceae [11][G-2] sp. HOT091¹ Streptococcus constellatus ² Campylobacter rectus¹ Peptostreptococcaceae [11][G-4] sp. HOT369¹ Streptocuccus parasanguinis_II¹ Clostridiales[F-1][G-1] sp. HOT093¹ Peptostreptococcaceae [13][G-1] sp. HOT113¹ Synergistes G36¹ Desulfobulbus sp. HOT041³ Peptostreptococcus stomatis² Tannerella forsythia⁴ Dialister invisus¹ Porphyromonas endodontalis⁴ TM7 [G-1] sp. HOT346² Enterobacter cancerogenus¹ Porphyromonas gingivalis⁴ TM7 [G-1] sp. HOT349³ Eubacterium brachy² Prevotella buccae¹ TM7 [G-5] sp. HOT356² Eubacterium minutum¹ Prevotella denticola² TM7 [G-5] sp. HOT437¹ Eubacterium nodatum² Prevotella intermedia³ Treponema amylovorum¹ Eubacterium saphenum³ Prevotella melaninogenica¹ Treponema denticola⁴ Eubacterium yurii¹ Prevotella oralis¹ Treponema lecithinolyticum³ Filifactor alocis⁴ Prevotella oris¹ Treponema maltophilum³ Fretibacterium fastidiosum³ Prevotella oulorum¹ Treponema medium² Fretibacterium sp. HOT360⁴ Prevotella sp. HOT292¹ Treponema socranskii ⁴ Fretibacterium sp. HOT361² Prevotella sp. HOT526¹ Treponema sp. HOT230¹ Fretibacterium sp. HOT362¹ Prevotella tannerae² Treponema sp. HOT237³ Fusobacterium A71¹ Pseudoramibacter alactolyticus² Treponema sp. HOT246¹ Fusobacterium nucleatum ss animalis¹ (1 core) Rothia dentocariosa¹ (3 health) Treponema sp. HOT257¹ Fusobacterium nucleatum ss polymorphum¹ (1 health) Schwartzia sp. HOT129¹ Treponema sp. HOT490¹ Fusobacterium nucleatum ss vincentii¹ (2 core) Schwartzia sp. HOT132¹ unclassifi ed Clostridiales¹ Fusobacterium sp. HOT203¹ Schwartzia sp. HOT145² Johnsonella sp. HOT166¹ Schwartzia sp. HOT150¹

FIGURE 2. Health-associated, core and periodontitis-associated species of the subgingival microbiome. Green and red shapes show species with signifi cantly diff erent relative abundances in health and periodontitis. Data to construct this fi gure were extracted from our own studies15,17 and the studies of Griff en et al.14 and Kirst et al.16 Species supported by the four included high-throughput sequencing studies are underlined. Gray shape shows core species, which are those detected in a majority of the subjects surveyed and that did not change in relative abundance from health to periodontitis, according to Abusleme et al.15 and Hong et al.17 The number of studies that support the association of a species with each group is indicated as a superscript (maximum of four studies for green and red shapes and maximum of two studies from gray shape). In case of a disagreement, the species name was placed in both groups and the number of studies that support its placement in either group indicated.

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range of taxa, most of which remain community and that their presence is associated species can be detected in understudied from all biological likely important in the microbiome periodontitis and periodontitis-associated aspects. FIGURE 2 compiles data from shifts from health to periodontitis.15,17 species in health, albeit in low proportions the four high-throughput sequencing Interestingly, the most abundant core and at a lower frequency.14,15 Therefore, studies mentioned above and shows the species in both of our studies15,17 was periodontitis is associated with shifts in health-associated and periodontitis- Fusobacterium nucleatum, a Gram-negative the species that numerically dominate associated members of the subgingival anaerobe with a demonstrated ability subgingival communities rather than microbiome, that is, the species to interact physically via coaggregation caused by de novo colonization. Evidence that increase or decrease in relative with a diverse range of oral species.18 of the indigenous nature of periodontitis- abundance according to clinical status. F. nucleatum has also been shown to associated species was previously provided Most studies usually detect more metabolically support the growth of by more targeted analyses, which showed periodontitis-associated than health- periodontitis-associated taxa in several that colonization of periodontitis- associated species, as periodontitis in vitro investigations. In a continuous- associated species occurs early in life.21,22 communities are more diverse due to culture aerated polymicrobial community, A better understanding of microbiome a more even distribution, in terms of shifts from health to periodontitis needs percentages, of the species that form the to consider not only changes in species community. It is evident from FIGURE proportions, but also changes in biomass. 2 that the communities associated The development of Techniques such as 16S rRNA gene with health and periodontitis are targeted strategies to control sequencing can only reveal shifts in the complex entities and that a thorough subgingival plaque maturation relative proportions of species but do not understanding of this disease would measure changes in biomass of specifi c require a profound study of each may benefi t by focusing on taxa. To understand the evolution of community member and its interplay interspecies interactions the subgingival microbiome from this with other species and the host. involving core members. perspective, in one of our studies we Studies by our group also focused measured changes in the total bacterial on identifying those species that appear load from health to periodontitis using in most subjects and do not change real-time quantitative PCR.15 We found in their proportions from health to F. nucleatum had a positive infl uence that after a single pass of a curette, disease.15,17 We named these taxa, on the biomass of the Gram-negative 1,000 times more bacterial cells could depicted in FIGURE 2, the “core species” anaerobes P. gingivalis and Prevotella be recovered from periodontitis sites of the subgingival microbiome. Core nigrescens.19 Moreover, F. nucleatum (with probing depths (PD) equal to 5 species are bound to be metabolically can easily adapt to aerated conditions mm) in comparison to sites in healthy versatile as they are capable of thriving by metabolizing oxygen via enzymatic subjects (with PD <4 mm). These under the nutritional and environmental activities such as that of NADH oxidase, measurements were in close agreement conditions present in both health and thereby reducing the environment to with those previously reported by Moore periodontitis. Most important, core anaerobic levels in which P. gingivalis can and Moore using a cultivation approach.8 20 species are probably capable of synergistic thrive. F. nucleatum also generates CO2, This 3-log increase in biomass could interactions with health- and disease- which is subsequently metabolized by P. not solely be explained by the larger associated species as they successfully gingivalis.20 The development of targeted surface area of periodontal pockets, but grow with both groups. Also, they are strategies to control subgingival plaque is probably the result of a combination of able to grow well under various types maturation may benefi t by focusing on continued biofi lm accretion due to lack of community arrangements, as they interspecies interactions involving core of disruption by adequate hygiene and of are very prevalent despite variations in members, as they may have an essential increased community growth capacity. community composition from subject role facilitating microbial successions. The fl ow rate of gingival crevicular fl uid to subject. Because of this versatility, Although there are species strongly (GCF) into the sulcus has been shown we hypothesized that core species act as associated with health or disease, it is to gradually increase from health to metabolic cornerstones for the whole also important to highlight that health- gingivitis and to periodontitis.23,24 Since

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■ Health-associated species ■ Core species 108 ■ Periodontitis-associated species 10 7 ■ 48% subgingival communities rely on GCF Variable species 25% 17% as a nutritional source, communities 10% 6 in periodontitis may achieve higher 10 biomass due to the greater availability 10 5 of infl ammation-derived nutrients. The 58% shifts in composition of periodontitis- 10 4 25% 12% associated communities may also 5%

Bacterial cells 3 facilitate synergistic nutrient acquisition 10 activities. Enrichment cultivation 10 2 studies in serum and evaluations of the growth of periodontitis-associated 10 1 taxa in the presence of serum proteins suggest that increases in GCF fl ow rate 10 0 are likely to promote the enrichment Periodontal health Periodontitis of proteinase-rich taxa, which would in turn release peptides that could be FIGURE 3. Microbiome shifts from health to periodontitis depicting changes in total load of health-associated, utilized by themselves and by other core and periodontitis-associated species. Bars represent total load of each group in health and periodontitis. species in the community.25-28 Relative abundances for each group are also indicated by percentages above each bar. Percentages were Our group has also measured changes calculated from data tables generated by Abusleme et al.15 and Hong et al.17 Total loads were then calculated 15 in the total load of specifi c genera from assuming a 3-log change in biomass from health to periodontitis. Variable species were those not strongly associated with health or disease and not detected in most subjects. health to periodontitis.15 The health- associated genus Actinomyces was shown not to change its total biomass from health to disease. Therefore, its decreased in biomass from health to periodontitis role during periodontitis in terms of their relative proportion in periodontitis is and, therefore, decrease in terms of interaction with the immune system as the consequence of lack of growth as the their proportion. It should be noted their load is still signifi cant in disease. whole community matures and increases that although the load of certain species 16S rRNA gene sequencing studies in biomass. Actinomyces are, therefore, such as Actinomyces may not change as also revealed that microbiome shifts outcompeted during microbiome shifts. shown by qPCR, other health-associated occur in the whole mouth rather than In contrast, the total number of cells of species such as streptococci are likely to at specifi c tooth sites. Griffen et al.14 the core genus Veillonella, which in our proliferate with the whole community,15 showed that the communities of healthy study did not change in terms of relative and, therefore, the total load of health- sites in subjects with periodontitis proportions from health to periodontitis, associated species also increases from are depleted of health-associated taxa was shown to be higher in disease. The health to periodontitis. In contrast, and have increased proportions of dynamics of microbiome evolution from FIGURE 3 shows that core species periodontitis-associated species when health to periodontitis in terms of total increase their biomass from health to compared to sites in periodontitis- number of bacteria present at each stage disease in a much more dramatic way free individuals. An obvious clinical are shown in FIGURE 3. To construct than health-associated species, thereby implication from this fi nding is that this fi gure, we calculated the sum of preserving their proportion within treatment of periodontitis requires health-associated, core or periodontitis- the community. The most drastic a global, whole mouth approach. associated species under the healthy or increase in biomass, however, occurs Taking all these data together, it the periodontitis states, using relative for periodontitis-associated species, can be concluded that the subgingival abundance tables from our published which show a ~4-log increase becoming microbiome community evolves from studies.15,17 We then assumed a 3-log dominant in disease. This fi gure makes health to periodontitis by a process change in total biomass from health to it evident that not only periodontitis- of microbial succession, with the periodontitis. As FIGURE 3 shows, health- associated, but also health-associated and emergence of newly dominant species, associated species show the least changes core species, could have an important but without replacement of pioneer

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TABLE 3

Key Points Summarizing Microbiome Shifts From Health to Periodontitis

■ Community biomass increases by at least 3-log from health to periodontitis. ■ Microbiome diversity and, therefore, community complexity increases in periodontitis due to a more even distribution of species. implying greater nutrient availability ■ Health-associated species are those that occupy ~60 percent of the biomass in health and only ~10 and increased community productivity percent of the biomass in periodontitis. in disease. Also importantly, although ■ Periodontitis-associated species increase their biomass ~4-log from health to periodontitis. They communities from different subjects occupy 5 percent of the biomass in health and 50 percent of the biomass in periodontitis. ~ ~ harbored different species, the community ■ Microbiome shifts are not localized but involve to some degree all subgingival sites in the mouth of an metabolic activities were conserved among individual. subjects, showing there is redundancy ■ Microbiome shifts associated with gingivitis are distinct to those in periodontitis, with gingivitis possibly representing a transitional stage from health to disease. in terms of the species that can perform specifi c metabolic functions within a community. This study also showed that although the core species F. nucleatum did health-associated species, as we have health-associated and core species also not vary in abundance in shallow and deep previously proposed.15 TABLE 3 presents contributed to such increased functions, sites, it upregulated lysine fermentation a summary of key points regarding in accordance with the concept that to butyrate in the deeper pockets.31 This current knowledge on microbiome the whole community responds to the switch to butyrate suggests a change from shifts from health to periodontitis. changes in environmental conditions a more oxygenated to a more anaerobic that accompany pocket formation.29 One environment as pocket depth increased, Beyond 16S rRNA Sequencing: of the most widely upregulated functions in agreement with our previous work Understanding Changes in Community among species during periodontitis was which showed F. nucleatum switches its Function From Health to Periodontitis iron acquisition, suggesting that the fermentation end products from acetate via Metatranscriptomics ability to compete for this nutrient may under oxygenated conditions to butyrate Subgingival communities can be an important determinant of which in a more reduced environment.32 also be characterized via shotgun species are ultimately able to thrive as A recent study by Yost et DNA sequencing, which provides an biomass accumulates. Another widely al.33 evaluated the subgingival overall view of all genes or via RNA upregulated function in periodontitis metatranscriptome during chronic sequencing, which reveals all RNA was response to oxidative stress, which periodontitis progression comparing transcripts in a given community. A could be a consequence of increased subject-matched progressing and non- metatranscriptomic study (sequencing neutrophil presence in disease, as these progressing sites at two time points. This of all RNA transcripts in a community) cells utilize oxygen radical generation study revealed that communities from permits evaluation of community as a killing mechanism. Alternatively, nonprogressing sites were very stable in composition based solely on taxa that increased oxidative stress could also terms of their composition and metabolic are metabolically active. It also allows result from greater abundance in activities, contrary to communities from examination of changes in the metabolic periodontal pockets of proteinase- sites that lost attachment. Streptococci activities of specifi c species or metabolic rich species with the subsequent and other health-associated species such changes in the community as a whole. degradation of iron-containing serum as Actinomyces became less metabolically Metatranscriptomics has been used to proteins such as transferrin. Proteinases active during progression, while compare the subgingival microbiome stimulate bacterial growth via the Prevotella spp., Pseudomonas spp. and in health and periodontitis.29 This release of peptides and iron, but have the archaea Methanobrevibacter smithii, evaluation revealed that periodontitis also been shown to contribute to among others became more active. With communities have augmented biological oxygen radical generation when respect to the metabolic activities of processes related to fl agellar motility, targeting iron-containing proteins.30 the communities, the gene ontology peptide transport, iron acquisition, In another study, Jorth et al.31 (GO) terms of pathogenesis, response to beta-lactam degradation, lipid A compared the metatranscriptome of oxidative stress, ferrous iron transport, biosynthesis and cellular stress responses. shallow and deep pockets in subjects with protein secretion and growth, among Overexpression of these processes in aggressive periodontitis, showing that a others, became enriched in sites that periodontitis did not only occur in larger fraction of the microbial population broke down. The most striking fi nding periodontitis-associated species, but is metabolically active in deep pockets, of this study, however, was the large

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Gingivitis-associated species

Actinomyces dentalis1 Porphyromonas sp. HOT2791 Aggregatibacter sp. HOT5131 Prevotella denticola1 Bacteroidales [G-2] sp. HOT2741 Prevotella intermedia1 Campylobacter showae2 Prevotella loescheii1 Capnocytophaga leadbetteri1 Prevotella maculosa1 Capnocytophaga sp. HOT3321 Prevotella melaninogenica1 Species depleted in gingivitis Capnocytophaga sp. HOT8631 Prevotella micans1 Catonella morbi1 Prevotella oulorum2 Clostridiales [F-2][G-2] sp. HOT0751 Prevotella pallens1 1 Actinobaculum sp. HOT183 Clostridiales [F-2][G-2] sp. HOT0851 Prevotella saccharolytica1 1 Actinomyces naeslundii 1 1 1 Dialister invisus Prevotella salivae Actinomyces odontolyticus 2 1 Actinomyces sp. HOT1691 Fusobacterium nucleatum ss polymorphum Prevotella veroralis 1 1 Capnocytophaga gingivalis1 Gemella morbillorum Propionibacterium propionicum Capnocytophaga sputigena1 Granulicatella elegans1 Schwartzia sp. HOT135-145-148-1551 Corynebacterium durum2 Johnsonella ignava1 Selenomonas infelix1 Haemophilus parainfl uenzae2 Lachnoanaerobaculum saburreum1 Selenomonas noxia1 Kingella oralis1 Lachnoanaerobaculum umeaense1 Selenomonas sputigena1 Lautropia AP0091 Lachnospiraceae [G-3] sp. HOT1001 Solobacterium moorei1 1 Leptotrichia hongkongensis Lautropia A941 SR1 [G-1] sp. HOT3451 1 Neisseria elongata Leptotrichia buccalis1 Streptococcus anginosus1 1 Rothia aeria 1 1 2 Leptotrichia hofstadii Streptococcus cristatus Rothia dentocariosa 1 1 Streptococcus A951 Leptotrichia shahii Streptococcus oligofermentans 1 2 Streptococcus E141 Leptotrichia sp. HOT212 Tannerella sp. HOT286 Streptococcus mitis2 Leptotrichia sp. HOT2231 Tannerella sp. HOT8081 Streptococcus sanguinis1 Leptotrichia sp. HOT2251 TM7 [G-1] sp. HOT3461 Streptococcus sp. HOT0641 Leptotrichia sp. HOT4171 TM7 [G-1] sp. HOT3471 Streptococcus sp. HOT4231 Leptotrichia sp. HOT4631 TM7 [G-1] sp. HOT3481 Leptotrichia sp. HOT4981 TM7 [G-1] sp. HOT3491 Leptotrichia wadei1 TM7 7BB4281 Neisseria fl avescens1 Treponema socranskii1 Neisseria sp. HOT0181 unclassifi ed Comamonadaceae FX0061 Parvimonas micra1 uncultured Lachnospiraceae D00081 Peptococcus sp. HOT1671 uncultured Leptotrichia sp. 302H021 Peptostreptococcus stomatis1 uncultured Leptotrichia sp. 501D101 Porphyromonas catoniae2

FIGURE 4. Microbiome shifts in gingivitis. Graph shows species depleted (health-associated) and enriched (gingivitis-associated) during the health-to-gingivitis microbiome shift. Species in purple and orange shapes are those with a signifi cant change in relative abundance when comparing health and gingivitis, as reported by Kistler et al.35 and Huang et al.36 Superscripts indicate the number of studies that support their placement on each group (maximum of two studies). differences in the metabolic activities in baselines of sites that progressed. With These results suggest that since the of communities from baseline samples respect to the community metabolic community activities were different (before attachment loss occurred) of activities, biological processes such as prior to detectable tissue destruction, progressing and nonprogressing sites. A lipid A and peptidoglycan biosynthesis, the local microenvironment may also comparison of the species that were active response to oxidative stress, fl agellar have been different with increased in baseline samples showed that a great motility and amino acid, iron and infl ammation at these sites. Also, inter- number of species, belonging to all groups potassium transport were enriched in the species interactions and community as defi ned in this review, were more active baselines of sites that lost attachment.33 arrangements in these communities

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may have been distinct allowing greater two to three weeks.34 Signifi cantly, these Catonella morbi present in both groups overall community metabolism. shifts correlate with the appearance of (FIGURES 2 and 4). Gingivitis-associated Taking it all together, clinical signs of gingivitis. Recent studies species, however, appeared among those metatranscriptomic analyses of the using high-throughput sequencing of we designated as variable species when subgingival microbiome suggest that 16S rRNA genes in conjunction with health and periodontitis were compared the microbial community adapts to the experimental plaque accumulation (FIGURE 3). These species were not changing nutritional and environmental models agree with the classic cultivation strongly associated with health or with conditions in the gingival crevices. studies but defi ne, with greater accuracy, periodontitis and were not consistently Because biomass increases with disease, the molecular taxonomy of shifts during detected as they were not enriched probably due to greater availability of gingivitis onset.35,36 FIGURE 4 depicts the under either condition. Therefore, the carbon sources originating from GCF, species associated with the transition microbiome shift associated with gingival it is expected that competition for the from health to gingivitis according to infl ammation is unique and distinct to the scarcest resources also becomes fi erce and the studies of Kistler et al.35 and Huang health-associated microbiome and to the those species able to adapt and capture the et al.36 It is apparent from this fi gure that shift associated with tissue destruction. limiting nutrients (e.g., iron, which is a compound of low bioavailability) are those Proposing Gingivitis-Associated that ultimately thrive. Metatranscriptomic and Core Species as Mediators of studies, therefore, highlight the ecological The microbiome shift Transitions From Health to Periodontitis nature of microbiome shifts and the associated with gingival Our group recently explored whether importance of understanding community infl ammation is unique and subjects belonging to the same clinical adaptation to environmental pressures. category (health or disease) harbored distinct to the health-associated different microbiome subtypes, perhaps Subgingival Microbiome Shifts in microbiome and to the shift refl ecting different stages of dysbiosis.17 Gingivitis associated with tissue destruction. We found that communities from healthy Gingivitis is defi ned as the presence subjects tended to cluster into two groups, of infl ammation in the gingival tissues one large cluster L, with the majority of without loss of connective tissue subjects, and one small cluster, designated attachment and bone resorption. Because the microbiome becomes more complex S, with only a few subjects. Cluster gingivitis is thought to be a required in gingivitis with enrichment of a great L samples were enriched for health- step in the transition from health to variety of taxa, in accordance with an associated Streptococcus, Actinomyces, periodontitis, oral microbiologists increase in diversity as reported by high- Rothia, Lautropia, Bergeyella, Kingella and have also focused on understanding throughput sequencing.35,37 Moreover, Granulicatella species. In contrast, cluster S microbiome shifts under this condition. most of the species depleted in gingivitis samples were enriched for species from the Furthermore, because gingivitis can are characterized by their aerobic or genera Bacteroidales[G-2], Capnocytophaga, be induced in humans by oral hygiene facultative anaerobic metabolism, while Campylobacter, Corynebacterium, abstention and the condition resolves most enriched species are anaerobes, Fusobacterium, Leptotrichia, Parvimonas, after resumption of plaque control, the which suggests the formation of anaerobic Porphyromonas, Prevotella, Selenomonas, human experimental model of gingivitis microniches during biofi lm accretion. Tannerella and TM7[G-1], the majority has been repeatedly characterized.34-36 Most species enriched in gingivitis are of which are gingivitis-associated or core Classic studies performed via also Gram-negative. However, the species as defi ned in this review (FIGURES 2, 4 cultivation assessing plaque evolution at enriched in gingivitis are not the same and 5). Similarly, in Hong et al.17 we the gingival margin during oral hygiene as those associated with periodontitis. In explored the existence of community abstention show a shift from a dominance fact, out of the 69 gingivitis-associated types within periodontitis fi nding also a of Gram-positive cocci during the initial taxa shown in FIGURE 4, only 14 tendency for two clusters to form. One days of plaque accumulation to an increase species are also periodontitis-associated. periodontitis cluster (designated cluster in Gram-negative morphotypes including Gingivitis-associated species are also A) was enriched for species from the rods, fi laments and spirochaetes after not the same as core species, with only genera Campylobacter, Corynebacterium,

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FIGURE 5. Proposed model of temporal shifts in the subgingival microbiome from health to periodontitis with gingivitis-associated and core species as mediators of transitions. Pie charts depict species proportions as shifts occur from health to periodontitis. Green represents health-associated species, gray shows core species, red depicts periodontitis-associated species and yellow shows variable species, as defined in this review. Gingivitis-associated species are depicted in orange. Depicted proportion for gingivitis-associated species is hypothetical as the studies available did not have this information. Proportions of species in health and periodontitis pie charts are based on data from Hong et al.,17 who found two types of health (clusters L and S) and two types of periodontitis communities (clusters A and B), potentially representing temporal stages of progressive dysbiosis. Cluster L communities were enriched for health-associated species with respect to cluster S samples, which in turn were enriched for core and gingivitis-associated taxa. Cluster A samples were enriched for core and gingivitis-associated species with respect to cluster B, which, in turn, was enriched for species strongly associated with periodontitis. Cluster B taxa are positively correlated with periodontitis severity.17 Examples of species enriched at each stage are shown below the pie charts. Gingivitis-associated species appear in orange text, while core species appear in gray text to highlight the enrichment of these species types in clusters S and A. Text and arrows above the pie charts indicate the events mediating microbiome shifts from health to periodontitis with gingivitis as a transitional stage.

Fusobacterium, Leptotrichia, Prevotella, of sites involved) suggesting a later core species is an intermediate stage in Tannerella and TM7[G-1] species, many temporal stage in the disease process.17 the health-to-periodontitis microbiome of which are also gingivitis-associated The data discussed above, although shifts. As shown in FIGURE 5, a great or core species. In contrast, the second cross-sectional, suggests that the different number of species enriched in the health periodontitis cluster detected (cluster clusters represent a temporal progression. cluster S and in the periodontitis cluster B) was enriched with the red complex, In FIGURE 5 we have put together a A are also gingivitis-associated, with Filifactor alocis, Treponema spp., conceptual model of the microbiome also some core species. Indeed, whether Fretibacterium spp. and other species shifts from health to periodontitis, based gingivitis represents a transitional stage strongly associated with periodontitis. on the microbial clusters found by Hong between heath and periodontitis has Cluster B was also associated with greater et al.17 In this model, we propose that the been a matter of debate. However, very periodontitis severity (greater number enrichment of gingivitis-associated and few studies have been properly designed

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to address this question. One notable serum-derived nutritional resources.26 are likely to mediate tissue destruction.4 exception is the study by Tanner et al.,38 The species that fl ourish under gingivitis, After initial periodontal breakdown, who evaluated progression of periodontitis however, need to have relative tolerance periodontitis-associated species increase in subjects with very mild levels of to oxygen as the sulcus depth is shallow in biomass, but gingivitis-associated disease in their whole mouth. This study at this stage. In this respect, gingivitis- and core species are still important found a positive correlation between associated anaerobes such as F. nucleatum community components.38 Profound baseline levels of clinical gingivitis (as and Prevotella melaninogenica have been dysbiosis is detected once pockets have measured by gingival index and bleeding shown to have increased tolerance to formed with a fi nal microbiome transition on probing) with mean changes in PD, oxygen than periodontitis-associated in which red complex species, Filifactor suggesting gingivitis indeed mediates species such as P. gingivalis and Treponema alocis, Fretibacterium spp., among others, the health-to-disease transition. spp., which fl ourish at a later stage once outcompete other taxa. As more sites are In the model proposed in FIGURE 5, pockets form.20,40 During the health-to- compromised in a subject, the greater the each cluster represents a temporal stage gingivitis transition the community also chances of detecting these profoundly with examples of species enriched at each needs to adapt to the new infl ammatory dysbiotic communities representing the stage shown at the bottom of the fi gure. end-stage of microbiome shifts.17 As The potential interactions between the in health and gingivitis, interspecies microbiome and the host driving these interactions are probably essential to arrive transitions are shown at the top of FIGURE In clinical health, the to the stage of fi nal dysbiosis and maintain 5. In clinical health, the microbiome microbiome of most subjects the stability of these communities. of most subjects is enriched for health- is enriched for health- associated species such as Rothia aeria Intraspecies Populations Shifts Are and Actinomyces spp. (cluster L). Cluster associated species such Also Likely Important in Health-to- S possibly represents a transitional as Rothia aeria and Disease Transitions stage of subclinical dysbiosis, in which Actinomyces spp. (cluster L). Microorganisms belonging to the the microbiome is shifting toward the same species exhibit a certain degree gingivitis state. This initial shift is of genetic diversity and, therefore, a mediated by biofi lm accretion due to species constitutes a heterogeneous inadequate oral hygiene as demonstrated environment. With greater biomass comes population.42 Rapid succession of by the experimental gingivitis studies.2,35,36 increased competition and, therefore, bacterial types within a species has been During this phase, the growth of the interspecies interactions, some of them seen to occur during the initial stages community would be mostly driven by antagonistic, would continue to infl uence of biofi lm formation in the oral cavity.43 interspecies interactions because oral taxa which species succeed as shifts occur. It is expected that during microbiome depend on each other for growth under The host defenses are also likely to exert shifts, intraspecies populations will the nutritional conditions present in great environmental pressure at this stage also undergo selection as some cells the gingival sulcus.39 If biofi lm accretion selecting for those species more resistant will be better able to adapt to certain is not disrupted, microbial successions to the actions of antimicrobial host environments depending on their occur with the rise of gingivitis-associated effectors such as complement and iron- attributes. The presence of a capsule species, which are likely to depend on sequestering lactoferrin, which have been in certain P. gingivalis strains may the prior growth of health-associated shown to be increased in gingivitis.41 represent a good example of a virulence taxa. The increased biomass and these FIGURE 5 then proposes that gingivitis attribute mediating intraspecies shifts. initial shifts in species abundances would communities are likely to set the stage Genotypes similar to encapsulated P. represent a higher load of microorganisms, for a subsequent shift characterized by gingivalis strains seem to predominate especially Gram-negative, and their the rise of periodontitis-associated taxa. in periodontal pockets, while non- products to which the host responds. The events that lead to the transition encapsulated genotypes predominate The resultant infl ammation probably from gingivitis to periodontitis are in health.44 The presence of a capsule exerts an environmental pressure that less clear but it seems subversion and has been shown to confer P. gingivalis selects for species effective at utilizing dysregulation of the immune responses the ability to avoid neutrophil killing

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and, therefore, encapsulated P. gingivalis needed. Moreover, microbiome shifts Chambers DW. Relationship between the percentage of are better at subverting immune are likely to depend on interspecies subgingival spirochetes and the severity of periodontal disease. 45 J Periodontol 1982; 53(9): 550-6. surveillance. However, encapsulated metabolic interactions. Communities 7. Socransky SS, Haff ajee AD, Cugini MA, Smith C, Kent RL Jr. P. gingivalis strains are not effi cient at in health are probably needed for Microbial complexes in subgingival plaque. J Clin Periodontol forming biofi lms,46 which may explain gingivitis-associated taxa to fl ourish, 1998; 25(2): 134-44. 8. Moore WE, Moore LV. The bacteria of periodontal diseases. why the encapsulated genotypes are not while gingivitis-associated and core taxa Periodontol 2000 1994; 5: 66-77. the predominant ones found prior to are likely to set the stage for the rise of 9. Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR. pocket formation, when retention via periodontitis-associated species. The Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A 1985; biofi lm formation would be important. metabolic interactions mediating these 82(20): 6955-9. Thus, in the healthy shallow sulcus, successions, however, have not been 10. Fox GE, Stackebrandt E, Hespell RB, Gibson J, Maniloff non-encapsulated P. gingivalis may clearly defi ned. It can be concluded that J, Dyer TA, Wolfe RS, Balch WE, Tanner RS, Magrum LJ, Zablen LB, Blakemore R, Gupta R, Bonen L, Lewis BJ, Stahl have an advantage over encapsulated the development of novel strategies DA, Luehrsen KR, Chen KN, Woese CR. The phylogeny of types in terms of retention. Non- to treat or prevent periodontitis needs prokaryotes. Science 1980; 209(4455): 457-63. encapsulated types, however, stimulate to take into account environmental 11. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, Sahasrabudhe A, Dewhirst FE. Bacterial diversity host responses in a much more effective modifi cation and interspecies in human subgingival plaque. J Bacteriol 2001; 183(12): way and are, therefore, contained by dependencies. The identifi cation of 3770-83. the immune system. Once a pocket species with key metabolic roles in 12. Kumar PS, Griff en AL, Moeschberger ML, Leys EJ. Identifi cation of candidate periodontal pathogens and forms, non-encapsulated genotypes the transitional communities may benefi cial species by quantitative 16S clonal analysis. J Clin can more easily thrive in the sulcus as represent a step forward as these species Microbiol 2005; 43(8): 3944-55. part of the loosely adherent plaque, could represent better therapeutic 13. Dewhirst FE, Chen T, Izard J, Paster BJ, Tanner AC, Yu WH, Lakshmanan A, Wade WG. The human oral microbiome. J while avoiding phagocytes. The P. targets than periodontitis-associated Bacteriol 2010; 192(19): 5002-17. gingivalis case exemplifi es why the species such as the red complex, whose 14. Griff en AL, Beall CJ, Campbell JH, Firestone ND, Kumar study of intraspecies population dominance seems to be the ultimate PS, Yang ZK, Podar M, Leys EJ. Distinct and complex bacterial profi les in human periodontitis and health revealed by 16S shifts is also essential, although little result of tissue destruction. ■ pyrosequencing. ISME J 2012; 6(6): 1176-85. attention has been paid to it. 15. Abusleme L, Dupuy AK, Dutzan N, Silva N, Burleson ACKNOWLEDGMENT JA, Strausbaugh LD, Gamonal J, Diaz PI. The subgingival Work related to this manuscript was funded by grants R01 microbiome in health and periodontitis and its relationship with Conclusion DE021578 and R21 DE023967 from the National Institute community biomass and infl ammation. 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Periodontal Res 2015 Oct;50(5):637-49. doi: 10.1111/ 25. ter Steeg PF, van der Hoeven JS, de Jong MH, van Munster jre.12244. Epub 2014 Nov 29. THE CORRESPONDING AUTHOR, Patricia I. Diaz, DDS, PhD, can be PJJ, Jansen MJH. Modelling the gingival pocket by enrichment 42. Kilian M, Frandsen EV, Haubek D, Poulsen K. The etiology reached at [email protected]. of subgingival microfl ora in human serum in chemostats. of periodontal disease revisited by population genetic analysis. Microb Ecol Health Dis 1988; 1: 73-84. Periodontol 2000 2006; 42: 158-79. 26. ter Steeg PF, Van der Hoeven JS, de Jong MH, van 43. Palmer RJ Jr., Diaz PI, Kolenbrander PE. Rapid succession Munster PJ, Jansen MJ. Enrichment of subgingival microfl ora within the Veillonella population of a developing human oral on human serum leading to accumulation of Bacteroides biofi lm in situ. J Bacteriol 2006; 188(11): 4117-24. species, Peptostreptococci and Fusobacteria. Antonie Van 44. Igboin CO, Griff en AL, Leys EJ. Porphyromonas gingivalis Leeuwenhoek 1987; 53(4): 261-72. 27. Grenier D, Imbeault S, Plamondon P, Grenier G, Nakayama K, Mayrand D. Role of gingipains in growth of Porphyromonas gingivalis in the presence of human serum albumin. Infect Immun 2001; 69(8): 5166-72. 28. Grenier D, Mayrand D, McBride B. Further studies on the degradation of immunoglobulins by black-pigmented Bacteroides. Oral Microbiol Immunol 1989; 4: 12-18. CDA Cares turns compassion 29. Duran-Pinedo AE, Chen T, Teles R, Starr JR, Wang X, Krishnan K, Frias-Lopez J. Community-wide transcriptome of into community in action. the oral microbiome in subjects with and without periodontitis. ISME J 2014; 8(8): 1659-72. 30. Goulet V, Britigan B, Nakayama K, Grenier D. Cleavage of human transferrin by Porphyromonas gingivalis gingipains promotes growth and formation of hydroxyl radicals. Infect CDA Cares Immun 2004; 72(8): 4351-6. 31. Jorth P, Turner KH, Gumus P, Nizam N, Buduneli Stockton N, Whiteley M. Metatranscriptomics of the human oral microbiome during health and disease. MBio 2014; 5(2): Oct. 15–16, e01012-14. 2016 32. Diaz PI, Zilm PS, Rogers AH. The response to oxidative stress of Fusobacterium nucleatum grown in continuous culture. San Joaquin FEMS Microbiol Lett 2000; 187(1): 31-4. County Fairgrounds 33. Yost S, Duran-Pinedo AE, Teles R, Krishnan K, Frias-Lopez J. Functional signatures of oral dysbiosis during periodontitis 1658 S. Airport Way progression revealed by microbial metatranscriptome analysis. Stockton, CA 95206 Genome Med 2015; 7(1): 27. An estimated 10 million Californians experience 34. Theilade E, Wright WH, Jensen SB, Loe H. Experimental barriers to dental care, which compromises their gingivitis in man. II. A longitudinal clinical and bacteriological investigation. J Periodontal Res 1966; 1: 1-13. total health and their opportunities. A new 35. Kistler JO, Booth V, Bradshaw DJ, Wade WG. Bacterial smile can enhance employment outcomes, community development in experimental gingivitis. PLoS One improve quality of life and, for many, restore 2013; 8(8): e71227. 36. Huang S, Li R, Zeng X, He T, Zhao H, Chang A, Bo dignity. Making a difference in the lives of our C, Chen J, Yang F, Knight R, Liu J, Davis C, Xu J. Predictive neighbors, that’s what moves us. modeling of gingivitis severity and susceptibility via oral microbiota. ISME J 2014; 8(9): 1768-80. To volunteer or donate, please visit 37. Park OJ, Yi H, Jeon JH, Kang SS, Koo KT, Kum KY, Chun J, Yun CH, Han SH. Pyrosequencing Analysis of Subgingival cdafoundation.org/stockton Microbiota in Distinct Periodontal Conditions. J Dent Res 2015; 94(7): 921-7. 38. Tanner AC, Kent R, Jr., Kanasi E, Lu SC, Paster BJ, Sonis

JULY 2016 435 You are the protector of the smile. You enable people to laugh without shame, eat their favorite foods and experience the dignity of aging with grace. That’s why this association tirelessly advocates for the profession and stands up for those in need of care. Because the world is a better place when people are smiling, and that’s thanks to you.

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® caries

CDA JOURNAL, VOL 44, Nº7

Understanding Caries From the Oral Microbiome Perspective

Anne C.R. Tanner, BDS, PhD; Christine A. Kressirer, PhD; and Lina L. Faller, PhD

ABSTRACT Dental caries is a major disease of the oral cavity with profound clinical signifi cance. Caries results from a transition of a healthy oral microbiome into an acidogenic community of decreased microbial diversity in response to excessive dietary sugar intake. Microbiological cultivation, molecular identifi cation, gene expression and metabolomic analyses show the importance of the entire microbial community in understanding the role of the microbiome in the pathology of caries.

AUTHORS

Anne C.R. Tanner, Christine A. Kressirer, he term microbiome is causing disease and are not exogenous BDS, PhD, is a dental PhD, is a microbiologist generally attributed to pathogens as described for the classical microbiologist at the Forsyth at the Forsyth Institute in Joshua Lederberg1 who Koch’s Postulates. A second reason for Institute in Cambridge, Cambridge, Mass. Dr. Mass. Dr. Tanner earned Kressirer earned bachelor’s defi ned it as signifying “the this change in perspective came from her bachelor of dental degrees in biochemistry ecological community of the realization that there are benefi cial surgery and a doctoral and microbiology from Tcommensal, symbiotic and pathogenic members of the oral microbiota and degree from the University Colorado State University microorganisms that literally share that an understanding of health and of London. She was and a doctoral degree in our body space and have been all but disease required knowledge of all of awarded honorary degrees pharmaceutical biology from Umeå University in from Ludwigs Maximilian ignored as determinants of health and the microorganisms that share our Umeå, Sweden and the University in Munich. disease.” Since the late 19th century bodies, not just a select few pathogens. University of Connecticut. Confl ict of Interest when Robert Koch published his famous Confl ict of Interest Disclosure: None reported. postulates, the study of infectious The Human Microbiome Project Disclosure: None reported. disease and medical microbiology The Human Genome Project Lina L. Faller, PhD, is a bioinformatics analyst have focused primarily on individual provided a deep understanding of the at the Forsyth Institute in pathogens as causative agents of disease. more than 20,000 protein-coding genes Cambridge, Mass. Dr. This approach was highly successful for that underlie the functioning of the Faller earned a bachelor’s diseases such as cholera, tuberculosis human organism.2 Investigators who degree in computer science and anthrax, but in the past 20 years, participated in the Human Genome and master’s and doctoral degrees from Boston it has become apparent that focusing Project realized that it was not possible University’s bioinformatics on a single organism as the pathogen is to fully understand human physiology, graduate program. inappropriate for polymicrobial diseases health or disease without a similar Confl ict of Interest such as caries and periodontitis. This project to understand the microbes Disclosure: None reported. change in perspective came in part that inhabit us. The vision for the because the organisms involved in these Human Microbiome Project (HMP) was diseases, such as Streptococcus mutans articulated in a white paper published and Porphyromonas gingivalis, are present in 2007.2 The National Institutes in most individuals at low levels without of Health (NIH) project followed

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the next year with the key goals of Study of Bacteria Associated With Caries Based on cultural analysis, the caries- determining the microbial composition associated biofi lm was shown to include of the microbiome in 15 body sites in Early Culture Studies a wide range of bacterial species, with men and 18 in women from about 300 Prior to the 1980s, bacteria were principal suspected caries pathogens healthy adults and to determine the identifi ed in culture studies by growing including acidogenic streptococci, complete genomes of 3,000 human- them in petri dishes on bacterial media particularly S. mutans, Actinomyces, associated bacterial species. More and characterizing phenotypic traits such Bifi dobacterium and Lactobacillus species.22,27-31 than 200 papers have been published as Gram stain reaction, motility, aerobic describing the results of the HMP and or anaerobic growth, fermentation of Molecular Methods Expand the Caries- a major summary of its fi ndings was various carbohydrate, end products of Associated Microbiota published in 2012.3 Body sites studied fermentation and enzyme activities. Early Starting in the 1980s, the means for included nine from the oral cavity: oral microbiology studies in humans, identifying and classifying bacteria underwent subgingival, supragingival, keratinized including those of Loesche and Syed, a radical change from using cultural methods gingivae, hard palate, tongue dorsum, Hardie and Bowden and Gibbons and to using molecular methods. Molecular buccal mucosa, palatine tonsils, throat methods are based on gene sequences to and saliva. A major fi nding from these characterize and identify bacteria, most studies is that each of the oral sites frequently using the 16S rRNA gene that has a slightly different microbiome.4 Starting in the 1980s, the codes for a smaller subunit of the ribosome. Information on oral bacteria, their means for identifying and This approach was pioneered by Carl Woese taxonomy and genomic information is classifying bacteria underwent who recognized the importance of the 16S publicly available in the Human Oral rRNA gene for determining phylogeny Microbiome Database (homd.org).5 a radical change from using and used it to revise the tree of life for all cultural methods to using living organisms.32 In every environment Clinical Signifi cance molecular methods. examined, molecular methods revealed Dental caries is a worldwide a vast array of bacteria that had not been health concern with a very uneven previously identifi ed or described. Many of distribution of affected individuals. Caries the newly recognized bacteria had not been disproportionally affects socially and van Houte who isolated and named identifi ed using the prevailing methods economically disadvantaged populations.6 more than 100 oral species that set the of strain identifi cation using biochemical In the United States, costs for dental stage for studies on the microbiological tests, or had not been cultivated and so services were $84.4 billion in 2014 cause of caries.13-16 The approaches used were invisible in previous cultivation-based (National Health Expenditures, 2014),7 of to associate bacteria with caries were studies. Examination of the species present in which, caries restorative care comprised those of Koch and Pasteur, trying to the oral cavity using 16S rRNA methodology a substantial component. While dental link caries to a single pathogen. The was pioneered by the Relman and Dewhirst/ caries affects all ages, it can be particularly initial link between bacteria, notably Paster laboratories.33,34 The oral cavity is now devastating when it affects the primary Streptococcus mutans, Lactobacillus and known to have approximately 700 species teeth in young children.8,9 Treating early Actinomyces species and dental caries, identifi ed using 16S rRNA methods.5 These childhood caries (ECC)10 in children with came from observations in experimental studies have provided a solid understanding multiple affected teeth is diffi cult and animal models from investigators that of which bacteria are present in the entire frequently involves care under general included Jordan, Fitzgerald and Keyes, oral cavity and which are present at specifi c anesthesia.11 In older adults caries is Bowden, van Houte and Caulfi eld.17-21 sites as described below for dental caries. frequently associated with exposed root Cultivation studies linked S. mutans with Based on the extensive knowledge base surfaces resulting from gingival recession caries in humans22 and demonstrated for bacteria colonizing the supragingival and from medication-induced low salivary transmission of S. mutans in humans plaque and teeth, many molecular studies fl ow. This can compromise an individual’s from mother to child.23-25 Investigators have been performed to understand which ability to eat leading to malnutrition and showed an increased risk of caries bacteria are associated with different expediting mortality in the elderly.12 the younger a child was infected.26 types and stages of dental caries.

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A. White spot lesions and initial caries

S. mutans (C,M) Actinomyces gerensceriae (M) Veillonella parvula (M) S. sobrinus (M) Scardovia wiggsiae (M) Veillonella species (C,M) S. salivarius (M) Scardovia inopinata (M) Selenomonas sputigena (M) Streptococcus parasanguinis (M) Bifidobacterium species (M) Prevotella denticola (M) Non-mutans Streptococcus spp. (C) Atopobium parvulum (M) Prevotella species (M) Granulicatella elegans (M) Dialister invisus (M) Lactobacillus gasseri (M) Solobacterium moreii (M) Lactobacillus species (C)

B. Dentinal caries and tubule infection

S. mutans (C,M) Actinomyces israelii (C) Scardovia wiggsiae (C,M) S. sanguinis (C) Actinomyces odontolyticus (C) Scardovia inopinata (C) S. salivarius (C) Actinomyces naeslundii (C) Parascardovia denticolens (C) Lactobacillus rhamnosus (C,M) Rothia dentocariosa (C,M) Bifidobacterium dentium (C,M) Lactobacillus acidophilus (C) Propionibacterium acidifaciens (C,M) Bifidobacterium species (C,M) Lactobacillus paracasei (C) Olsenella profusa (C) Veillonella parvula (M) Lactobacillus fermentum (C) Olsenella species (M) Veillonella dispar (M) Lactobacillus plantarum (C) Atopobium species (M) Veillonella spp. (M)

Lactobacillus casei (M) Pseudoramibacter alactolyticus (M) Fusobacterium nucleatum (M) L. gasseri/johnsonii (M) Atopobium genomospecies C1 (M) Prevotella melaninogenica (M) Candida albicans (C)

C. Root caries

S. mutans (C,M) Propionibacterium species (C,M) Bifidobacterium dentium (C) S. mitis (C) Atopobium species (M) Bifidobacterium breve (C) S. intermedius (C) Olsenella species (M) Bifidobacterium species (C) S. sanguinis (C) Pseudoramibacter alactolyticus (M) Selenomonas Lactobacillus species (C,M) Propionebacterium acidifacians (M) Prevotella multisaccharivorax (M) Actinomyces israelii (C) Scardovia inopinata (C) Enterococcus faecalis (M) Actinomyces species (C,M) Scardovia wiggsiae (C) Parascardovia denticolens (C)

FIGURE 1. Principal species in caries-associated microbiomes. The major species detected by culture (C) or molecular (M) approaches. The diagrams illustrate position of lesions into enamel (white), dentin (orange), pulp (red). A = white spot initial lesions, B = dentin caries, C = root caries. Source information from references cited in the text.

Types of Caries and Their Microbiota healthy enamel surrounding them. WSLs Veillonella species are commonly There are many ways to subdivide are an early sign of a site at risk for caries observed in initial lesions and it has been types or stages of caries, but here we will lesion development in both primary and suggested that they support growth of highlight three types: White spot lesions secondary dentitions. Cultivation35,36 and streptococci by using lactic acid as an or early caries in the enamel, dentin molecular37-40 studies have identifi ed the energy source and facilitating the growth caries where the lesion has penetrated the major species involved, which are shown of acidogenic species.38 Even acid-tolerant enamel into the dentin, and root caries, in panel A of FIGURE 1. It is a consistent species show reduced growth at pH levels which occurs on exposed root surfaces of observation that S. mutans is not detected below pH 4.5 and benefi t from members cementum or dentin. FIGURE 1 illustrates in all white spot, or indeed all other types of the microbial community that help these types of caries and lists the major of carious lesions, whether assayed by maintain a more neutral pH environment. bacteria associated with these lesions. cultivation or molecular methods. Other consortia of acidogenic bacteria including Dentin Caries White Spot Lesions nonmutans streptococci and Actinomyces As caries extends into and eventually White spot lesions (WSLs) are species are, however, present when S. through the enamel, it reaches into noncavitated precarious lesions in enamel mutans is not detected. These studies have dentin and dentinal tubules. As the and represent an early stage of tooth also shown that bacterial species diversity dentin breaks down a cavity is formed demineralization. Due to mineral loss, is reduced in caries compared with healthy that, without intervention, will WSLs have an opaque and chalky-white sites, suggesting an acidic environment ultimately progress into the pulp and appearance when compared with the suppresses acid-sensitive bacteria.41 nerve fi bers. The key culture studies

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100% ■ **Streptococcus mutans Species ■ *S. vestibularis salivarius 90% of dentin caries in adults are from ■ *V. atypica dispar parvula Wade42 and Hunter43 and in children ■ *Streptococcus sobrinus 44 ■ *S. parasanguinis nonsignifi cant species from severe ECC from Beighton 80% and Tanner45 laboratories. Molecular ■ **Lachnospiraceae oral taxon 107 studies have included focus on ■ *Capnocytophaga gingivalis Lactobacillus and Prevotella species in 70% ■ *Cardiobacterium hominis adults46 and childhood caries.37,38 The ■ *Campylobacter rectus dentin microbiota is characterized by ■ *Streptococcus cristatus 60% a more anaerobic microbiome than ■ *Capnocytophaga granulosa caries-free sites or initial lesions. ■ *Granulicatella adiacens In addition to S. mutans there are 50% ■ *Streptococcus AY020 increasing proportions of acidogenic ■ *Corynebacterium matruchotii acid-tolerant gram positive rod species ■ **Kingella oral taxon 499 45 40% in Actinomyces, the Bifi dobacterium ■ *Neisseria elongate 47 family (Bifi dobacterium, Scardovia, etc.) ■ *Kingella denitrifi cans 48 and Lactobacillus species. Increased 30% ■ **Corynebacterium durum detection of S. mutans in dentinal caries ■ **Abiotrophia defective in adolescents with abundant biofi lm ■ *Streptococcus sanguinis 20% and extensive caries compared with ■ *Neisseria fl ava mucosa pharyngis adolescents with routine dental care ■ *S. mitis pneumoniae infantis suggests that S. mutans infection may 10% be population dependent.49 Increasing * = p<0.05, ** p<0.01 proportions of the highly acid- (after false discovery rate adjustment) tolerant Bifi dobacterium/Scardovia and 0% Lactobacillus taxa in advanced lesions HealthyWhite Intact Cavitated fi t with the expanded ecological plaque spot enamel hypothesis50 described below. The dentin microbiota also includes signifi cant 45,51 proportions of more acid sensitive, FIGURE 2. Bacterial communities of progressing ECC. Communities identifi ed to species from health (caries-free but acid-producing species in Prevotella, children) and three sites of ECC children: intact enamel, WSL and cavities. The microbiome composition changed Capnocytophaga and Selenomonas. from health to disease with increases in proportions of S. mutans and Veillonella in the microbiome. From: Gross EL, Caries deep in dentin may not be Beall CJ, Kutsch SR, Firestone ND, Leys EJ, Griff en AL. Beyond streptococcus mutans: Dental caries onset linked to at an extremely low pH52 suggesting multiple species by 16s rRNA community analysis. PLoS One 2012;7:e47722. Top fi ve taxa are caries-associated and the rest are associated with health. that these Gram-negative anaerobes can play an active role in caries. The key species identifi ed in these studies have been associated with root caries Culture Compared With Molecular are shown in panel B of FIGURE 1. as well as yeast and Streptococcus Analyses species (panel C in FIGURE 1).30,53-56 It Comparisons between molecular Root Caries has been observed that the microbial methods and anaerobic culture Root caries affects dentin exposed composition of carious lesions vary showed many similarities in the taxa from gingival recession or periodontal between subjects more so than in WSLs identifi ed, but also some key differences. surgery in adults and the elderly. Risk and dentinal caries, thus there is a less Molecular analyses allow for greater factors include reduced salivary fl ow characteristic microbiota consistently depth of samples and more species can from medications or disease (Sjögren’s observed. The major species associated be detected, especially fastidious and syndrome), sugar-sweetened, syrupy with root caries are listed in panel C diffi cult-to-grow species and species not medications and frequent intakes of of FIGURE 1 and includes many of the yet cultured. Cultural methods, however, cariogenic foods. Gram-positive rods same species found in dentinal caries. enhanced detection of other species

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including S. mutans, Actinomyces and formation and progression. This model Example of Caries Microbiome Bifi dobacterium/Scardovia species.45,57 incorporates the concept that various Community Changing With The reasons these species were missed stages of dental caries are associated Progression in many molecular studies is that the with different microbial communities. FIGURE 2 illustrates the changes that bacterial cells are diffi cult to break While some interspecies occur in the oral biofi lm community in open for DNA release and their DNA interactions can enhance virulence subjects with caries at different stages is not recognized by standard primer in an environment,62-65 cooperation of disease, sampled to represent caries and adapters used for 16S rRNA among species may also reduce progression. The data for this fi gure amplifi cations42,58 and pyrosequencing.59 pathogenicity. The concept of Koch’s came from studies that combined culture Many of the “not yet cultured species” Postulates focusing on one species for and molecular approaches to examine inferred by molecular methods were one disease has been reevaluated in severe ECC progression37,38 (FIGURE 2). found as isolates in investigator’s strain the era of microbial community-based Advanced dentin lesions were found collections when these collections diseases. Byrd and Segre suggest that to harbor a new Bifi dobacterium species were characterized using 16S rRNA the presence of pathogens may not (an early report of Scardovia wiggsiae sequencing.42,45 Thus, fi ndings from in caries), S. mutans and veillonellae38 molecular and cultural methods have in addition to previously uncultivated proved complementary in identifying species. In another study using the the bacteria associated with caries. Findings from molecular same clinical design, Actinomyces and nonmutans streptococci with higher Caries Models and cultural methods have levels of S. mutans were identifi ed in Models of the roles of bacteria proved complementary in association with increasing lesion depth.37 in caries development including identifying the bacteria Species in higher levels in dentin than nonspecifi c, specifi c60 and ecological S. mutans included Bifi dobacterium plaque hypotheses61 have been based associated with caries. dentium, nonmutans streptococci, and on data from culture studies. The Veillonella, Atopobium, Propionebacterium extended ecological hypothesis of and Lactobacillus species. In children’s Takahashi and Nyvad incorporated permanent teeth, dentin-colonizing three stages in caries development.50 necessarily equate to disease because species included S. mutans, Lactobacillus The fi rst dynamic stability stage had a of colonization resistance.66 In this spp. Propionebacterium and Atopobium dominance of nonmutans streptococci scenario, the community confers an species in contrast to bifi dobacteria in and Actinomyces that produced mild and ameliorating impact on pathogenicity. primary teeth.37 In adults, carious lesion infrequent acidifi cation that was not In caries, acidity produced by S. mutans, development was also associated with a associated with net tooth mineral loss. for example, can be counterbalanced shift in microbiome composition with The second acidogenic stage followed by ammonia production from arginine increasing proportions of S. mutans, acid-induced adaptation or selection deiminase or urease activity from other several Lactobacillus species, Scardovia to those nonmutans streptococci and species.67 Thus, while investigators inopinata and Rothia dentocariosa.70 Actinomyces species that could lower might aim to defi ne the most likely These studies indicated differences the pH. This led to moderate and and most frequently active pathogens, in microbiota with lesion progression more frequent acid formation likely their detection may not defi nitely from caries-free sites to WSL and to associated with initial enamel or dentin indicate disease. This is illustrated cavities, with increasing detection of S. (root caries) demineralization. The by the observations that despite mutans, lactobacilli and bifi dobacteria fi nal aciduric phase was characterized strong associations of S. mutans to with disease, although S. mutans was by increased acid-induced adaptation ECC by culture,28,68 and of S. mutans not found in all carious sites. These or selection with a microbial increase and S. sobrinus to ECC using PCR data illustrate that in the caries in mutans streptococci and other methods,69 the mutans streptococci process, the entire microbiome is aciduric bacteria. Clinically, this have not proved universally reliable reshaped with changes in the relative translated to tooth mineral loss, lesion in caries risk assessment.9 proportion of essentially all species.

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pH range 4.5–5.0 5.0–5.5 5.5–6.0 6.0–6.5 >6.5 Phylum ■ Bacteria unclassifi ed ■ Actinobacteria ■ Bacteroidetes ■ Firmicutes ■ Fusobacteria ■ Proteobacteria ■ SR1 ■ Spirochaetes ■ TM7

Genus ■ Atopobium ■ Streptococcus ■ Afi pia ■ Olsenella ■ Dialister ■ Acinetobacter ■ Actinomyces ■ Shuttleworthia ■ Under 0.2% ■ Propionibacterium ■ Eubacterium ■ Parascardovia ■ Oribacterium ■ Bifi dobacterium ■ Lachnospiraceae(unc.) ■ Rothia ■ Peptostreptococcaceae(unc.) ■ Bifi dobacteriaceae(unc.) ■ Filifactor ■ Scardovia ■ Parvimonas ■ Corynebacterium ■ Fusobacterium ■ Prevotella ■ Leptotrichia ■ Lactobacillus ■ Sphingomonas ■ Pseudoramibacter ■ Escherichia

Species ■ Actinomyce sp.(unc.) ■ Lactobacillus crispatus ■ Propionibacterium acidifaciens ■ Lactobacillus rhamnosus ■ Bifi dobacterium dentium ■ Lactobacillus salivarius ■ Parascardovia denticolens ■ Streptococcus downei ■ Atopobium rimae ■ Shuttleworthia satelles ■ Atopobium sp.(unc.) ■ Pseudoramibacter alactolyticus ■ Olsenella profusa ■ Dialister invisus ■ Prevotella denticola ■ Fusobacterium nucleatum ■ Prevotella multisaccharivorax ■ Sphingomonas sp.(unc.) ■ Prevotella oris ■ Under 1% ■ Prevotella sp.(unc.)

FIGURE 3. Microbiome profi le of dentinal caries by lesion pH. Community profi les by relative abundance at phylum, genus and species levels in dentin with pHs ranging from pH 4.5 to pH > 6.5. The fi gure illustrates high proportions of Firmicutes (phylum) Lactobacillus (genus) and L. crispatus at lowest pHs. There was a marked increase in microbial diversity detected at the higher pHs, refl ecting the reduction of species in the less acidic environment. From: Kianoush N, Adler CJ, Nguyen KA, Browne GV, Simonian M, Hunter N. Bacterial profi le of dentine caries and the impact of pH on bacterial population diversity. PLoS One 2014a;9:e92940.

Example of Caries Microbiome three panels present the changes in at higher levels at pH 5.5-6 (p = 0.042).48 Community Changing With pH microbial profi le looking at phylum-, Higher bacterial counts were observed of Lesion genus- and species-level identifi cations. in surface and superfi cial dentinal layers The effect of pH on the dentin Increased Lactobacillus levels, but not S. compared to deeper dentinal layers, microbiome was recently explored in a mutans or S. sobrinus, correlated with a whereas surface and shallow lesion layers study of the association between lesion lower pH (FIGURE 3). Lactobacilli were were more acidic than deeper in dentin.52 activity, pH and depth.52 The results of associated with pH 4.5-5.0 (p = 0.0003) Lower microbial diversity was observed this study are shown in FIGURE 3. The whereas Prevotella species were detected with increased dentin acidity.71 The

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more acidic dentin had high proportions is another approach to examine what the importance of knowing microbiome of Lactobacillus and Atopobium (in the bacteria are doing. These new meta- activity as well as composition and the Actinobacteria) species whereas in less approaches as applied to dental caries were value of studying health in addition to acidic lesions the microbiome was more reviewed in depth by Nyvad, Crielaard, disease particularly in relation to ammonia diverse. This fi nding is consistent with Mira, Takahashi and Beighton.72 production to counteract dietary-associated the observation that the caries microbiota The association between low pH pH drops in the oral microbiome. can be less diverse than that of caries-free and caries was established by Stefan in sites, suggesting suppression of the acid 194473 and repeated for initial caries in Analysis of the Spatial Organization sensitive bacteria in the low pH of active van Houte’s laboratory.36 Metagenomic of Plaque lesions. Different pH profi les from surface analysis indicated increased mixed-acid Cultural and molecular methods have to deeper layers of dentin was observed fermentations in caries compared with indicated which bacteria are present in between individuals,48 which likely caries-free biofi lms,74 In contrast, there in carious lesions but not their spatial refl ects different microbial complexes was more enzyme activity in caries-free organization. Microscopy can show the associated with lesions of varying activity. than caries subjects75 suggesting that acid spatial arrangement of bacteria, but usually The pH of active lesions at pH 5.5 ± 0.3 without identifi cation of the bacteria was lower than that of arrested lesions imaged. Fluorescence in situ hybridization pH 6.1 ± 0.02 (p < 0.05)52 in keeping (FISH) uses fl uorescently labeled gene with the acid theory of dental caries. The major way researchers probes to identify specifi c bacteria. In look at what cells (either FIGURE 4, FISH confocal microscopy was Understanding Not Only Who’s There, bacterial or host) are doing used to examine bacteria in association But What They Are Doing with occlusal caries.82 The microbiome The Human Microbiome Project was is to look at gene expression in cavities included Lactobacillus and interested in fi nding out not only which or which proteins the cells are Bifi dobacterium species (FIGURES 4I–J) bacteria were present at various body actively making. and both these acidogenic, acid tolerant sites, but also to begin to understand what bacterial groups were observed invading they were doing at the molecular level. tubules (FIGURES 4K–L) in keeping with Answering this biological question is the the microbiota identifi ed from dentin next major step in understanding the from caries-associated microbiomes could caries. S. mutans was detected in all carious microbiomes role in human health and be destroying enzyme activity needed sites (FIGURE 4H), whereas bifi dobacteria disease. The major way researchers look to maintain health. Two critical biofi lm were observed only in active lesions further at what cells (either bacterial or host) enzymes associated with health76 are urease confi rming the importance of these species are doing is to look at gene expression and arginine deiminase that produce in caries pathogenicity. Other species or which proteins the cells are actively ammonia and can raise the biofi lm pH.67,77,78 examined were not directly associated with making. This type of study is called Clinically, caries-free children and adults lesion sites, for example Fusobacterium transcriptomics and is performed by had higher arginine deiminase levels than species (FIGURE 4B) were observed in a sequencing the messenger RNA. In all those with caries.76,79 Using a combination zone between lesion-associated biofi lm cells, genes are coded in the DNA. Genes of metabolomics and metatranscriptomics and a looser surface biofi lm mass. This that the cell needs are transcribed from several new pathways were found for observation concurs with the hypothesis DNA into messenger RNA. The RNA counterbalancing low pH and data that fusobacteria can bridge tooth message is translated on the ribosome suggested that ammonia production from adherent and other bacteria in gingival where amino acids are assembled arginine deiminase might be active in biofi lms.83 Veillonella species (FIGURE 4A) into proteins. Metatranscriptomics is initial lesions, whereas urease activity might and Streptococcus mitis (FIGURE 4C) were simply looking at the transcriptome balance the acidic microbiota of more observed in surface biofi lm layers suggesting of many organisms at a time, such as advanced disease.80 Study of metabolomics that they did not have a direct cariogenic all the bacteria in a plaque sample. in twins noted higher levels of arginine role in lesions. Overall, these observations Metabolomics is used to determine the and lysine in caries-free compared with concur with the major species detected metabolic activity of the microbiome and caries children.81 These studies indicate by molecular and cultural assays. This

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FIGURES 4. Bacteria in situ in carious lesions. A–L are images of biofi lm, including caries-associate biofi lms on occlusal surfaces, stained with situ hybridization (FISH) probes. The red stain is general bacterial stain. Specifi c species or genus probes are for Streptococcus yellow-green (A–E, J); Veillonella purple-magenta (A); Fusobacterium purple-magenta (B); S. mitis turquoise (C); Actinomyces purple-magenta (D); Lactobacillus purple-magenta (F, J, L); Bifi dobacterium purple-magenta (G,K); S. mutans yellow- green (H,J). From: Dige I, Gronkjaer L, Nyvad B. Molecular studies of the structural ecology of natural occlusal caries. Caries Res 2014;48:451-460.

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phylogenetic resolution should greatly REFERENCES increase our understanding of the bacteria- 1. Lederberg J. Of Men and Microbes. NPQ 2003;20(3):52-55. 2. Turnbaugh PJ, Ley RE, Hamady M, et al. The human bacteria and bacteria-host interactions in microbiome project. Nature 2007;449(7164):804-10. caries and other states of health and disease. 3. Human Microbiome Project C. A framework for human microbiome research. Nature 2012;486(7402):215-21. 4. Segata N, Haake SK, Mannon P, et al. Composition of FIGURE 5. The complex oral microbiome. Conclusions the adult digestive tract bacterial microbiome based on seven Combinatorial labeling and spectral imaging Dental caries manifests in several mouth surfaces, tonsils, throat and stool samples. Genome Biol fl uorescence in situ hybridization (CLASI-FISH) of clinical forms with differences in the 2012;13(6):R42. supragingival dental human plaque. The image composition of the microbiome based 5. Dewhirst FE, Chen T, Izard J, et al. 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The fi nal pH of bacteria process: Ecological perspectives. J Dent Res 2011;90(3):294-303. 71. Kianoush N, Adler CJ, Nguyen KA, et al. Bacterial profi le comprising the predominant fl ora on sound and carious human root 51. Svensater G, Larsson UB, Greif EC, Cvitkovitch DG, Hamilton of dentine caries and the impact of pH on bacterial population and enamel surfaces. J Dent Res 1996;75(4):1008-14. IR. Acid tolerance response and survival by oral bacteria. Oral diversity. PLoS One 2014;9(3):e92940. 31. Marsh PD, Featherstone A, McKee AS, et al. A microbiological Microbiol Immunol 1997;12(5):266-73. 72. Nyvad B, Crielaard W, Mira A, Takahashi N, Beighton D. study of early caries of approximal surfaces in schoolchildren. J Dent 52. Kuribayashi M, Kitasako Y, Matin K, et al. Intraoral pH Dental caries from a molecular microbiological perspective. Caries Res 1989;68(7):1151-4. measurement of carious lesions with qPCR of cariogenic bacteria to Res 2013;47(2):89-102. 32. Woese CR, Fox GE. Phylogenetic structure of the prokaryotic diff erentiate caries activity. J Dent 2012;40(3):222-8. 73. Stephan RM. Intraoral hydrogen-ion concentrations associated domain: The primary kingdoms. Proc Natl Acad Sci U S A 53. Brailsford SR, Shah B, Simons D, et al. The predominant aciduric with dental caries activity. J Dent Res 1944;23(4):257-66. 1977;74(11):5088-90. microfl ora of root-caries lesions. J Dent Res 2001;80(9):1828-33. 74. Belda-Ferre P, Alcaraz LD, Cabrera-Rubio R, et al. The oral 33. Paster BJ, Boches SK, Galvin JL, et al. Bacterial diversity in human 54. Ikebe K, Imazato S, Izutani N, et al. Association of salivary metagenome in health and disease. ISME J 2012;6(1):46-56. subgingival plaque. J Bacteriol 2001;183(12):3770-83. Streptococcus mutans levels determined by rapid detection system 75. Yang F, Ning K, Chang X, et al. Saliva microbiota carry caries- 34. Kroes I, Lepp PW, Relman DA. Bacterial diversity within the human using monoclonal antibodies with prevalence of root surface caries. specifi c functional gene signatures. PLoS One 2014;9(2):e76458. subgingival crevice. Proc Natl Acad Sci U S A 1999;96(25):14547- Am J Dent 2008;21(5):283-7. 76. Nascimento MM, Browngardt C, Xiaohui X, et al. The eff ect 52. 55. Mantzourani M, Fenlon M, Beighton D. Association between of arginine on oral biofi lm communities. Mol Oral Microbiol 35. Boyar RM, Thylstrup A, Holmen L, Bowden GH. The microfl ora Bifi dobacteriaceae and the clinical severity of root caries lesions. 2014;29(1):45-54. associated with the development of initial enamel decalcifi cation Oral Microbiol Immunol 2009;24(1):32-7. 77. Kleinberg I. A mixed-bacteria ecological approach to below orthodontic bands in vivo in children living in a fl uoridated- 56. Hashimoto K, Sato T, Shimauchi H, Takahashi N. Profi ling of understanding the role of the oral bacteria in dental caries causation: water area. 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Critical evaluation of two oral metaproteome reveals potential biomarkers for caries disease. Microbiol 2008;46(4):1407-17. primers commonly used for amplifi cation of bacterial 16S rRNA Proteomics 2015;15(20):3497-507. 38. Becker MR, Paster BJ, Leys EJ, et al. Molecular analysis of genes. Appl Environ Microbiol 2008;74(8):2461-70. 80. Edlund A, Yang Y, Yooseph S, et al. Meta-omics uncover bacterial species associated with childhood caries. J Clin Microbiol 59. Schulze-Schweifi ng K, Banerjee A, Wade WG. Comparison temporal regulation of pathways across oral microbiome genera 2002;40(3):1001-09. of bacterial culture and 16S rRNA community profi ling by clonal during in vitro sugar metabolism. ISME J 2015;9(12):2605-19. 39. Torlakovic L, Klepac-Ceraj V, Ogaard B, et al. Microbial analysis and pyrosequencing for the characterization of the 81. Foxman B, Srinivasan U, Wen A, et al. Exploring the eff ect community succession on developing lesions on human enamel. J dentine caries-associated microbiome. Front Cell Infect Microbiol of dentition, dental decay and familiality on oral health using Oral Microbiol 2012;4(1):e16125. 2014;4:164. metabolomics. Infect Genet Evol 2014;22:201-7. 40. Tanner AC, Sonis AL, Lif Holgerson P, et al. White-spot lesions 60. Loesche WJ. Clinical and microbiological aspects of 82. Dige I, Gronkjaer L, Nyvad B. Molecular studies of the structural and gingivitis microbiotas in orthodontic patients. J Dent Res chemotherapeutic agents used according to the specifi c plaque ecology of natural occlusal caries. Caries Res 2014;48(5):451-60. 2012;91(9):853-8. hypothesis. J Dent Res 1979;58(12):2404-12. 83. Kolenbrander PE, Palmer RJ, Jr., Rickard AH, et al. Bacterial 41. Gross EL, Beall CJ, Kutsch SR, et al. Beyond Streptococcus mutans: 61. Marsh PD. Dental plaque as a biofi lm and a microbial interactions and successions during plaque development. Dental caries onset linked to multiple species by 16S rRNA community community — implications for health and disease. BMC Oral Health Periodontol 2000 2006;42:47-79. analysis. PLoS One 2012;7(10):e47722. 2006;6 (Suppl 1:S14). 84. Valm AM, Mark Welch JL, Rieken CW, et al. Systems-level 42. Munson MA, Banerjee A, Watson TF, Wade WG. Molecular 62. Shen S, Samaranayake LP, Yip HK. In vitro growth, acidogenicity analysis of microbial community organization through combinatorial analysis of the microfl ora associated with dental caries. J Clin and cariogenicity of predominant human root caries fl ora. J Dent labeling and spectral imaging. Proc Natl Acad Sci USA Microbiol 2004;42(7):3023-29. 2004;32(8):667-78. 2011;108(10):4152-7. 43. Martin FE, Nadkarni MA, Jacques NA, Hunter N. Quantitative 63. Filoche SK, Anderson SA, Sissons CH. Biofi lm growth of 85. Mark Welch JL, Rossetti BJ, Rieken CW, Dewhirst FE, Borisy GG. microbiological study of human carious dentine by culture and real- Lactobacillus species is promoted by Actinomyces species and Biogeography of a human oral microbiome at the micron scale. Proc time PCR: Association of anaerobes with histopathological changes in Streptococcus mutans. Oral Microbiol Immunol 2004;19(5):322-6. Natl Acad Sci USA 2016;113(6):E791-800. chronic pulpitis. J Clin Microbiol 2002;40(5):1698-704. 64. Shen S, Samaranayake LP, Yip HK. Coaggregation profi les 44. Marchant S, Brailsford SR, Twomey AC, Roberts GJ, Beighton of the microfl ora from root surface caries lesions. Arch Oral Biol THE CORRESPONDING AUTHOR, Anne C.R. Tanner, BDS, PhD, can be D. The predominant microfl ora of nursing caries lesions. Caries Res 2005;50(1):23-32. reached at [email protected].

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Uncultured Members of the Oral Microbiome

William Wade, BSc, PhD; Hayley Thompson, BSc, PhD; Alexandra Rybalka, BSc, PhD; and Sonia Vartoukian, BDS, FDS, PhD

ABSTRACT Around one-third of oral bacteria cannot be cultured using conventional methods. Some bacteria have specifi c requirements for nutrients while others may be inhibited by substances in the culture media or produced by other bacteria. Oral bacteria have evolved as part of multispecies biofi lms, and many thus require interaction with other bacterial species to grow. In vitro models have been developed that mimic these interactions and have been used to grow previously uncultivated organisms.

AUTHORS

William Wade, BSc, Alexandra Rybalka, he human mouth is heavily in pathogenesis and contribution to PhD, is a professor of BSc, PhD, is a postdoctoral colonized by microorganisms antimicrobial resistance is unknown, oral microbiology at scientist with interests in with all of the different types which is why there is substantial Queen Mary University oral microbiology and the represented: bacteria, archaea, interest in culturing uncultivated of London with interests in development of novel model the characterization of the systems to study uncultured fungi, protozoa and viruses. This bacteria and subjecting them to detailed human microbiome and oral bacteria. Treview will focus on the bacteria because phenotypic and genomic analysis. development and evaluation Confl ict of Interest of their importance in the common dental The aim of this review is to list of novel strategies for the Disclosure: None reported. diseases, dental caries and periodontal the uncultivated members of the oral prevention and treatment diseases, and because the phenomenon microbiome, discuss the reasons why some of microbiome-associated Sonia Vartoukian, BDS, disease. FDS, PhD, is a specialist of unculturability has been extensively bacteria are diffi cult to culture in vitro Confl ict of Interest periodontist and lecturer in investigated in bacteria. It has long and describe recent advances in culturing Disclosure: None reported. oral microbiology at Queen been realized that not all bacteria that previously uncultivated oral bacteria. Mary University of London. can be seen under the microscope can Hayley Thompson, BSc, Confl ict of Interest be cultured in the laboratory. An early Defi nition of “Unculturability” PhD, is a postdoctoral Disclosure: None reported. scientist with interests in oral estimate of oral bacterial culturability Clearly all bacteria that can be microbiology, the virulence was that only around half could be detected on Earth have grown at some factors of pathogenic grown.1 Recent advances in culture time. Culturability is, therefore, a relative bacteria and uncultured have modifi ed this estimate so that it is term and dependent on the conditions oral bacteria. now considered that around two-thirds used to encourage growth in a particular Confl ict of Interest 2 Disclosure: None reported. of oral bacteria can be cultivated. experiment. A distinction should also be Although uncultivated bacteria cannot made between growth in monoculture be grown on commonly used laboratory and as part of a mixed community. The media, they clearly compete well in explosion in culture-independent studies the bacterial communities found in has revealed huge numbers of novel the mouth and many are associated bacterial taxa, the majority of which with oral disease. At present, their role cannot be identifi ed as belonging to

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Bergeyella HOT-900 Bergeyella HOT-907 Bergeyella HOT-322 Bergeyella HOT-931 Bergeyella HOT-422 Bergeyella zoohelcum Bergeyella HOT-319 Capnocytophaga HOT-901 Capnocytophaga ochracea previously cultivated and characterized Capnocytophaga HOT-903 species. This does not mean, however, Capnocytophaga HOT-334 that all of these taxa cannot be cultured. Capnocytophaga HOT-902 Indeed, a comprehensive cultural analysis of the microbiota of severe early childhood Bacteroidetes [G-6] HOT-516 caries revealed 45 species-level taxa that, Porphyromonas HOT-285 at that time, had not been cultivated.3 Porphyromonas asaccharolytica In addition, it is often forgotten that Porphyromonas HOT-275 in microbiome surveys, only DNA is Porphyromonas HOT-277 detected, not living cells, and the detection Porphyromonas HOT-930 of an organism’s DNA does not necessarily Tannerella forsythia mean that the organism was viable at Tannerella HOT-808 the time of sampling. Additionally, Tannerella HOT-916 DNA extraction and PCR reagents are Bacteroidetes [G-7] HOT-911 frequently contaminated with DNA from Bacteroidetes [G3] HOT-899 environmental bacteria and can make Bacteroidetes [G-3] HOT-281 up a signifi cant proportion of amplicon Bacteroidetes [G-3] HOT-503 libraries, particularly when samples are Bacteroidetes [G-3] HOT-280 taken from sites with low bacterial levels.4 Bacteroidetes [G-3] HOT-436 A practical defi nition of unculturability Bacteroides fragilis will be used for this review. An organism Alloprevotella HOT-308 will be regarded as uncultivated if there Alloprevotella HOT-914 are no reports that it has been grown Alloprevotella HOT-912 in previous cultivation studies. Alloprevotella HOT-913 Alloprevotella tannerae Uncultured Oral Bacteria Prevotella HOT-315 When culture-independent methods Prevotella HOT-515 were fi rst used to study the composition Prevotella HOT-526 of the oral microbiota and compared to Prevotella HOT-304 cultural analyses of the same samples, Prevotella HOT-942 it was clear that a substantial number Prevotella HOT-301 of bacterial taxa could not be readily Prevotella HOT-309 cultured.5,6 The Human Oral Microbiome Prevotella HOT-443 Database (HOMD, homd.org)7 lists Prevotella HOT-396 the bacteria found in the mouth. Many species-level taxa have yet to be named Prevotella melaninogenica and are, therefore, assigned human Prevotella HOT-305 oral taxon (HOT) numbers. HOMD Prevotella HOT-293 release 13.2 includes 210 species-level Prevotella HOT-292 taxa that have yet to be cultured. Many Prevotella HOT-300 uncultivated taxa belong to genera whose Bacteroidetes [G-4] HOT-509 members are predominantly cultivable. Bacteroidetes [G-5] HOT-507 Because still relatively few oral bacteria Bacteroidetes [G-5] HOT-505 have been cultured and identifi ed by 0.05 Bacteroidetes [G-5] HOT-511 16S rRNA gene sequence analysis, it is FIGURE 1. Phylogenetic tree showing uncultivated species-level oral taxa within the phylum Bacteroidetes. Tree prepared possible that these taxa are cultivable but by the neighbor-joining method from a distance matrix constructed using the Jukes-Cantor algorithm and an alignment of representative strains have not yet been 998 bases. Sequences representing type species of relevant genera are included for reference and colored red.

448 JULY 2016 CDA JOURNAL, VOL 44, Nº7 Selenomonas HOT-388 Selenomonas HOT-478 Selenomonas HOT-936 Selenomonas HOT-937 Selenomonas HOT-501 Selenomonas HOT-134 Selenomonas HOT-442 Selenomonas sputigena Mitsuokella HOT-521 Mitsuokella multiacida Veillonellaceae [G-1] HOT-132 Veillonellaceae [G-1] HOT-150 Veillonellaceae [G-1] HOT-148 Veillonellaceae [G-1] HOT-135 Veillonellaceae [G-1] HOT-918 Veillonellaceae [G-1] HOT-145 Veillonellaceae [G-1] HOT-483 encountered. Thus, the common genera Dialister HOT-119 Actinomyces, Prevotella, Streptococcus Dialister pneumosintes and Veillonella all include such taxa. Veillonella HOT-917 Veillonella parvula In contrast, many groups of Megasphaera HOT-123 uncultured taxa cluster in deep Megasphaera HOT-841 branches of the phylogenetic tree Megasphaera elsdenii Paenibacillus HOT-048 with no or few cultivated neighbors. Paenibacillus polymyxa FIGURE 1 shows a phylogenetic tree Erysipelothrix rhusiopathiae of the uncultivated members of the Erysipelotrichaceae [G-1] HOT-904 Erysipelotrichaceae [G-1] HOT-905 phylum Bacteroidetes. It can be seen Peptococcus HOT-168 that Bacteroidetes genera G-3 and G-7 Peptococcus niger comprise a branch of six uncultured Syntrophomonadaceae [VIII][G-1] HOT-435 Ruminococcus fl avefaciens species-level taxa of which Bacteroidetes Ruminococcaceae [G-1] HOT-075 [G-3] HOT-281 is the most commonly Ruminococcaceae [G-2] HOT-085 detected taxon, while Bacteroidetes Ruminococcaceae [G-3] HOT-366 Ruminococcaceae [G-3] HOT-381 genera G-4 and G-5 constitute another Clostridiales [F-1][G-1] HOT-093 deep branch with four uncultivated Clostridiales [F-1][G-2] HOT-402 taxa. Similarly, the phylum Firmicutes Peptoniphilaceae [G-2] HOT-790 includes a number of deep-branching Peptoniphilus asaccharolyticus Peptostreptococcaceae [XI][G-7] HOT-922 lineages made up of uncultivated taxa Peptostreptococcus anaerobius (FIGURE 2). These include a major Peptostreptococcaceae [XI][G-4] HOT-103 branch of the Peptostreptococcaceae Peptostreptococcaceae [XI][G-4] HOT-369 Peptostreptococcaceae [XI][G-5] HOT-493 with seven uncultivated taxa from Peptostreptococcaceae [XI][G-1] HOT-383 fi ve genera, four uncultivated Peptostreptococcaceae [XI][G-2] HOT-091 taxa within the Ruminococcaceae Peptostreptococcaceae [XI][G-3] HOT-495 Peptostreptococcaceae [XI][G-3] HOT-950 and a number of uncultivated Lachnospiraceae [G-10] HOT-094 representatives of the Lachnospiraceae, Catonella HOT-164 Syntrophomonadaceae and Veillonellaceae. Catonella morbi Catonella HOT-451 The Fusobacteria phylum includes Lachnospiraceae [G-7] HOT-086 an uncultured branch consisting of Lachnospiraceae [G-7] HOT-163 Fusobacteria [G-1] with two taxa: Butyrivibrio HOT-080 Butyrivibrio HOT-455 HOT-210 and HOT-220, and one Butyrivibrio HOT-090 comprised of uncultured Leptotrichia Butyrivibrio fi brisolvens taxa HOT-212, HOT-215, HOT- Lachnospiraceae [G-9] HOT-924 217 and HOT-392. Interestingly, the Stomatobaculum HOT-910 Stomatobaculum longum majority of Leptotrichia taxa have yet to Stomatobaculum HOT-097 be cultured, but most branches of this Stomatobaculum HOT-373 genus include cultivated members. Johnsonella HOT-166 Johnsonella ignava Bdellovibrio HOT-039 represents Lachnospiraceae [G-2] HOT-088 a deep uncultured branch within the Lachnospiraceae [G-3] HOT-100 phylum Proteobacteria. Bdellovibrio are Lachnoanaerobaculum HOT-496 Lachnoanaerobaculum HOT-089 normally aerobic, predatory bacteria, Lachnoanaerobaculum HOT-083 feeding on other Gram-negative bacteria8 0.05 Lachnoanaerobaculum umeaense and it will be interesting to know if this FIGURE 2. Phylogenetic tree showing uncultivated species-level oral taxa within the phylum Firmicutes. Tree prepared taxon has a similar predatory lifestyle. by the neighbor-joining method from a distance matrix constructed using the Jukes-Cantor algorithm and an alignment A substantial number of spirochetes of 1,126 bases. Sequences representing type species of relevant genera are included for reference and colored red. have yet to be cultured. All oral

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spirochetes belong to the genus within candidate division OP11.17 They for organisms formerly described as TM7.25 Treponema, and of the 49 oral Treponema are widely distributed, being found in Saccharibacteria are found in a range taxa, only 14 have been cultured. In anaerobic habitats such as deep-sea of animals. In invertebrates, they particular, one branch of 10 taxa has no sediments, various extreme environmental have been detected in the microbiota cultivable representatives: HOT-250-256, sites, the cow rumen and the human associated with sponges and corals,26-28 HOT-508, HOT-517 and HOT-518. mouth.18 SR1 comprises two lineages, termites29-31 and nematodes.32 They The recently described phylum BH1 and BD2-14, with the former found form part of the intestinal microbiota Synergistetes9 includes a large number only in geothermal habitats. Human of mammals and appear to be a of uncultured taxa. Oral members oral SR1 representatives are found in consistent member of the mammalian of the phylum form two clusters, subgroup III of the BD2-14 lineage. gut microbiome, having been found denoted A and B.10 The majority of Three species-level taxa belonging to in mice,33,34 cattle,35-37 dogs,38 pigs,39 Cluster A taxa have yet to be cultured, the same genus-level taxon are found elephants, gazelle, bighorn sheep, while Cluster B includes the recently in the mouth: HOT-345, HOT-874 and takin, buffalo, bonobo and gorillas.40 described species Jonquetella anthropi HOT-875. Like GN02, SR1 is a rare In humans, Saccharibacteria have been and Pyramidobacter piscolens.11 detected in several habitats, including Until recently, the phylum Chlorofl exi the intestinal tract,41 skin,42 vaginal fl uid43 has had no cultivated representatives and oral cavity44-46 although they typically among the oral microbiota, although Saccharibacteria appear to make up less than 1 percent of the environmental relatives have been community at a given site. Saccharibacteria cultivated.12 Three strains of Anaerolineae be associated with oral appear to be associated with oral disease, bacterium HOT-439, an important disease, particularly those particularly those conditions associated taxon thought to serve as a biomarker conditions associated with a with a mature anaerobic biofi lm. For for periodontitis,13 have recently example, Paster et al.44 found 34 sequences been isolated from subgingival plaque mature anaerobic biofi lm. representing the Saccharibacteria division samples and found to grow with the among 2,522 cloned 16S rRNA genes help of Fusobacterium nucleatum.14 from the subgingival plaque of healthy Three oral phyla have no, or very subjects and patients with periodontal few, cultivated representatives: GN02, member of the oral microbiome, and disease, which were later identifi ed as oral TM7 and SR1. Candidate division typically makes up around 0.01 percent taxa HOT-346, HOT-347, HOT-349, 355 GN02 was fi rst described to comprise a of clone libraries. At this level, it will be and HOT-356. Of these, only HOT-346 group of sequences identifi ed in a study challenging to detect individual cells by was found in health, while HOT-356 of the Guerrero Negro hypersaline fl uorescence in situ hybridization (FISH) (represented by phylotype I025, now microbial mat.15 Three oral taxa are or attempt direct isolation. Three species- recognized to belong to HOT-356) was found: HOT-871, HOT-872 and level taxa of SR1 have been identifi ed associated with periodontitis, a fi nding HOT-873, representing two class- among the canine oral microbiome, confi rmed in a study using oligonucleotide level taxa. Interestingly, four related but were only detected when a specifi c probes specifi c for HOT-356, in which the taxa were identifi ed among the canine Bacteroidetes-TM7-SR1 primer was used.16 taxon was found in 50 percent of healthy oral microbiome.16 Little is known The TM7 candidate division was subjects and 83 percent of patients with regarding the genetic potential or named with reference to the Torf, mittlere periodontitis.47 The same trend was seen in functional capability of this group of Schicht, or peat, middle layer, in which a polymerase chain reaction (PCR)-based organisms, although its ubiquity, albeit was fi rst detected in a German peat bog.19 study, with HOT-356 being detected in 91 at low levels, suggests that it deserves Subsequently, members of this division percent of diseased sites and 71 percent to be the target of future studies. have been isolated from a wide range of of controls, although the difference was Sequences representing candidate environments including waste water and not statistically signifi cant.48 In refractory division SR1 were originally detected batch reactor sludges,20,21 fresh and sea periodontitis, Saccharibacteria HOT-346, in sediments from Sulphur River in water22-24 and soil. The name Candidatus HOT-356 and HOT-437 were detected Parkers Cave, and were fi rst classifi ed Saccharibacteria has recently been proposed in signifi cantly higher proportions than

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in patients whose periodontal treatment and parasitic relationship with a strain of Atmospheric conditions, particularly was successful or healthy controls in a Actinomyces odontolyticus.55 It would appear the presence or absence of oxygen, study using the Human Oral Microbe then that the Saccharibacteria isolates as well as the availability of CO2 are Identifi cation Microarray (HOMIM).49 studied thus far are only found in such obviously extremely important. Some Three Saccharibacteria phylotypes close associations with other bacteria. It bacteria require specifi c nutrients were found in oral samples collected was further reported that association with for growth. Methanosaeta species, for from subjects with halitosis and one TM7x caused the A. odontolyticus host example, are obligately acetotrophic; of them, HOT-352, was signifi cantly to change its morphology from relative hence, the addition of acetone to associated with the condition.45 short rods to fi laments,56 although the media, which is slowly converted to The culture of members of the division growth phase and natural morphological acetate, will promote the growth of Saccharibacteria has long been a goal. It variation of both partners in the these otherwise slow-growing species.58 was reported that Saccharibacteria had interaction requires further investigation. Sometimes the medium itself been successfully cultured after 50-day The TM7x genome was found to be can be toxic. It has been shown that aerobic incubation of low-nutrient solid small at 705 kb and completing lacking autoclaving agar-containing culture media, with microcolonies visible to the media in the presence of phosphate can naked eye.50 No further detail of this generate inhibitory levels of hydrogen isolation has been reported, however. peroxide, an effect that can be avoided 59 There have been a number of reports of Strictly anaerobic bacteria by replacing agar with gellan gum. the successful isolation of Saccharibacteria Oral bacteria typically live as part bacteria in mixed culture. For example, coexist with oxygen-consuming of a multispecies community in densely microcolonies of Saccharibacteria from and -tolerant species and packed biofi lms. Within the biofi lm, there soil were obtained using a soil substrate cooperate to protect each are a variety of gradients of nutrients, membrane system.51 After seven days signaling molecules and gases, due to the incubation, several morphotypes were other from atmospheric stress. diffusion patterns of these substances and detected by means of FISH with the the metabolic activity of neighboring TM7-905 probe, although no pure cultures bacteria, such that conditions for were obtained. Using the same method, individual cells, and groups of cells, can Abrams et al.52 reported the isolation of in amino acid biosynthesis capability, vary markedly.60,61 Despite the mouth microcolonies that included cells that perhaps explaining its need to parasitize being exposed to the atmosphere, about reacted positively with the TM7 probe, other bacteria. Small genomes are a half of oral bacteria are obligate anaerobes. but also others that were negative with the feature of a number of other Divisions As oral biofi lms develop, obligate aerobes specifi c probe but positive for a universal yet to be cultured, including SR1, OD1 and facultative anaerobes rapidly reduce bacterial probe. Rybalka53 found that and WWE 3,57 suggesting that a limited the local oxygen concentration; four Saccharibacteria could be isolated in mixed metabolic repertoire and dependence on days of plaque formation in vivo in culture with a variety of other species, association with other organisms may be two subjects produced a mean redox including Slackia exigua and Atopobium common features of phylum-level taxa potential at the tooth surface of –127 parvulum, but could not be isolated in with no or few cultivable representatives. mV.62 Strictly anaerobic bacteria coexist pure culture or even maintained as a with oxygen-consuming and -tolerant mixture for more than a few subcultures. Reasons for Unculturability species and cooperate to protect each The successful isolation of a pure If bacteria are able to grow in a other from atmospheric stress.63 culture of a Saccharibacteria phylotype was particular environment but we are unable In a similar way, there will be a reported from a sample of dental plaque to cultivate them in the laboratory, then concentration gradient within the biofi lm but a culture was not deposited with a clearly at a basic level we are unable for nutrients with their concentration culture collection.54 A Saccharibacteria to reproduce the conditions that they decreasing with increasing depth of strain successfully isolated and maintained need for growth. Understanding these biofi lm, while bacterial metabolic from saliva, TM7x, was an extremely small conditions is key to the cultivation of products will be increased. There can coccus found in an exclusive physical previously uncultivated organisms. be direct interactions between species

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TABLE

Summary of Recently Described Methods for the Cultivation of Difficult-to-Culture Bacteria Approach to cultivation Examples Reference number Media supplementation, Addition of supplements to media e.g., siderophores, N-acyl homoserine lactones or growth 14, 59, 68, 73, 74, 91, customization or modifi cation factors; design of media selective for specifi c bacterial taxa using the SMART method; modifi ed 92 media preparation methods/substitution of agar with gellan gum as gelling agent, to limit growth inhibition by hydrogen peroxide.

Modifi cation of growth conditions Modifi ed temperature, pH, O2 presence/absence, incubation time, gravity. 93, 94 Modifi cation of sample handling Dilution-to-extinction to achieve single-cell isolation; small inoculum for reduced microbial 77, 78 competition. Simulated natural environment Diff usion chamber incubated within the natural environment allowing passage of growth- 89, 95, 96 stimulatory chemical compounds across a membrane; hollow-fi ber membrane chamber for in situ cultivation in the natural environment; I-tip in situ cultivation device permitting inward diff usion of natural chemical factors. Microfl uidic device Encapsulation of subsets of the microbial community to form microdroplets that are exposed to 97, 98 signals or nutrients from external bacteria. Community culture and co-culture Bacterial culture facilitated by chemical components produced by the main bacterial 14, 99–103 community separated from the target organism by a membrane, transwell insert or a well within the media plate; growth of bacteria in consortia, followed by detection and enrichment of specifi c bacterial targets using colony hybridization; co-culture of bacterial strains with “helper” species on which they depend for provision of growth factors or for environment modifi cation. High-throughput methods I chip: A device comprised of hundreds of miniature diff usion chambers, within each of which 96,104 a single cell is cultured; hollow-fi ber membrane chamber device (see above) comprised of 48–96 chamber units.

with one using the end products of peptide, thought to be a signaling molecule Methods for Culture of Uncultured another for growth. For example, stimulated the growth of a previously Bacteria Veillonella species use lactate produced by uncultivated Psychrobacter strain.68 A number of approaches have been streptococci as a major carbon source.64 Resuscitation-promoting factor (Rpf) is taken in attempts to culture previously Other possibly important factors in a protein that was identifi ed as being able uncultured bacteria and these are growth-regulating interactions between to revive Micrococcus cells from dormancy.69 summarized in the TABLE. Perhaps the most bacteria are bacterial signaling molecules. Rpf is structurally similar to lysozyme70 and promising approach is the recognition Gram-negative bacteria communicate cleaves peptidoglycan. Rpf is, therefore, that bacteria in nature frequently live in by means of acyl homoserine lactones likely to generate peptidoglycan fragments multispecies biofi lms and are, therefore, in (AHLs),65 and Gram-positives use small from the cell walls of intact bacteria, chemical contact with other bacteria. If diffusible peptides,66 but both systems which might act as signaling molecules.71 chemical interactions can be maintained are primarily intraspecies. In contrast, Muropeptide fragments are known to have in vitro, then isolation of novel organisms autoinducer-2 (AI-2), the product of the signaling properties in Bacillus subtilis, where should be possible. For example, D’Onofrio luxS gene, has homologues in a wide variety they bind to PrkC, a serine/threonine et al.73 grew seawater sediment bacteria of organisms, both Gram-positive and kinase on the cell surface.72 A specifi c on agar plates in mixed culture at various Gram-negative and has been suggested to muropeptide, a disaccharide-tripeptide dilutions. Because disproportionally more act across taxonomic boundaries.67 Bacterial with a meso-diaminopimelic-acid residue, colonies were seen on plates that had signaling affects a number of functional typically found in Gram-positive bacteria been heavily inoculated, pairs of colonies aspects including expression of the biofi lm is necessary for this activity. Rpf then may growing within 2 cm of each other were phenotype, production of virulence factors function by producing muropeptides from subcultured and then grown together. and growth itself. Bacteria accustomed peptidoglycan with signaling, possibly Around 10 percent of these pairs showed to growing in biofi lms may thus require growth-stimulating properties. Further evidence of the growth of one organism the presence of exogenous signals for work is required to investigate if this is a being dependent on its pair. The growth growth. For example, a small, 5-amino acid general method of growth regulation. of many of the dependent isolates was

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stimulated not only by its co-culture which led to the successful isolation of in samples of biomass.79,80 Thus, even partner but also by Escherichia coli. A several previously uncultivated bacterial though an organism cannot be grown, panel of E. coli mutants was, therefore, strains, including Chlorofl exi taxon its morphology can be determined. This constructed and tested to determine the Anaerolineae bacterium HOT-439.14 has been successfully performed for TM7, identity of the substance produced by E. One method of achieving a pure Tannerella BU063 and Synergistetes cluster coli that was stimulating the growth of culture of a slow-growing organism is the A.10,21,18 An alternative labeling method the dependent isolates. Enterobactin, a dilution-to-extinction method whereby is to use antibodies if it is possible to siderophore, was found to be responsible dilution ensures that single cells are select an appropriate specifi c antibody. and adding siderophores to culture media placed in a growth medium and have Because antibody labeling, unlike DNA allowed a number of novel bacteria to grow. time to grow without being inhibited by probing, is nonlethal, it can be possible A novel system has been developed other bacteria.75,76 A high-throughput to obtain viable cells for culture, after that explores genomic data for information version of the method was successful in sorting as described above; in this way on specifi c carbon source requirements cultivating a number of novel strains fl uorescent antibodies have been used to and antimicrobial resistance of particular of the seawater organism SAR11 as obtain viable cells after sorting.82 Flow bacteria, leading to the development cytometry can be used to isolate single of highly selective media designed by cells from mixtures from which whole SMART — selective medium-design genome sequences can be obtained; this algorithm restricted by two constraints.74 A range of compounds with approach has been successfully used Using this method, the authors prepared siderophore activity have to sequence genomes of the health- “selective” media for fi ve plant-pathogenic been screened for their ability associated taxon related to Tannerella bacteria and demonstrated accurate forsythia, HOT-286 (BU063).83 selection for the target bacterial species to stimulate the growth of oral Mixed primary cultures frequently among a panel of 18 strains representing bacteria that are unable to include representatives of bacterial 10 species. The use of such systems grow in pure culture. species not yet cultured in isolation. may provide a rational basis for the Colony hybridization is a useful method development of novel culture media. for determining the location of specifi c A range of compounds with taxa on solid media.84 By membrane siderophore activity have been screened well as representatives of the abundant, blotting and the use of specifi c probes, for their ability to stimulate the growth but previously uncultured, SAR116 target organisms can be localized to of oral bacteria that are unable to grow clade.77 Dilution to extinction would specifi c regions of replica plates, allowing in pure culture.14 Growth of Prevotella appear to be a method most suited to their subculture and enrichment. This HOT-376 was more strongly stimulated by samples such as seawater where bacterial method was used to culture the fi rst the siderophore pyoverdines-Fe, than by a concentrations are relatively low and representative of Synergistetes cluster culture fi ltrate of its helper Fusobacterium the bacterial cells are found primarily in A: Fretibacterium fastidiosum from nucleatum (the positive control); to a lesser planktonic suspension and interactions subgingival plaque in periodontitis.85,86 extent, it was also stimulated by ferric between bacteria are, therefore, limited. The isolation of previously citrate, desferricoprogen, ferrichrome-Fe- The oral microbiome, conversely, is uncultivated bacteria may be a multi- free and salicylic acid. Likewise, growth of primarily made up of dense biofi lms stage process. Clearly, the agar plate is Fretibacterium fastidiosum of Synergistetes where this method may be less generally an alien environment for bacteria used cluster A was consistently stimulated applicable, although some novel taxa to living in biofi lms associated with by desferricoprogen, salicylic acid and have been recovered using this method.78 mammalian tissues. One approach has ferrichrome-Fe-free. Consequently, Culture-independent surveys have been to establish biofi lms in vitro, seeded media used for culture of heavily made available 16S rRNA gene sequences with natural inocula. The Calgary Diofi lm diluted samples of subgingival plaque, for the microbiomes studied. These Device, a microplate-based system with were supplemented with siderophores data can then be used to design specifi c plastic pegs coated with hydroxyapatite pyoverdines-Fe or desferricoprogen, or oligonucleotide probes which can be used to mimic the tooth surface, has been a neat suspension of subgingival plaque, in FISH to visualize uncultured bacteria successfully used to produce dental

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New approaches “core microbiome” of the microbial communities in the tonsils of 44. for isolation of previously uncultivated oral bacteria. Appl Environ healthy pigs. BMC Microbiol 2012;12:20. 59. Tanaka T, Kawasaki K, Daimon S, et al. A hidden pitfall Microbiol 2012;78(1):194-203. 40. Ley RE, Hamady M, Lozupone C, et al. Evolution of mammals in the preparation of agar media undermines microorganism 79. Amann RI, Ludwig W, Schleifer KH. Phylogenetic identifi cation and their gut microbes. Science 2008;320(5883):1647-51. cultivability. Appl Environ Microbiol 2014;80(24):7659-66. and in situ detection of individual microbial cells without 41. Krogius-Kurikka L, Kassinen A, Paulin L, et al. Sequence 60. Stewart PS, Franklin MJ. Physiological heterogeneity in cultivation. Microbiol Rev 1995;59(1):143-69. analysis of percent G+C fraction libraries of human faecal biofi lms. Nat Rev Microbiol 2008;6(3):199-210. 80. Amann RI, Stromley J, Devereux R, Key R, Stahl DA. bacterial DNA reveals a high number of Actinobacteria. BMC 61. Kolenbrander PE. Oral microbial communities: biofi lms, Molecular and microscopic identifi cation of sulfate-reducing Microbiol 2009;9:68. interactions and genetic systems. Annu Rev Microbiol bacteria in multispecies biofi lms. Appl Environ Microbiol 42. Gao Z, Tseng CH, Pei Z, Blaser MJ. Molecular analysis of 2000;54:413-37. 1992;58(2):614-23. human forearm superfi cial skin bacterial biota. Proc Natl Acad 62. Kenney EB, Ash MM Jr. Oxidation reduction potential of 81. Zuger J, Luthi-Schaller H, Gmur R. Uncultivated Tannerella Sci U S A 2007;104(8):2927-32. developing plaque, periodontal pockets and gingival sulci. J BU045 and BU063 are slim segmented fi lamentous rods of 43. Fredricks DN, Fiedler TL, Marrazzo JM. Molecular Periodontol 1969;40(11):630-3. high prevalence but low abundance in infl ammatory disease- identifi cation of bacteria associated with bacterial vaginosis. N 63. Bradshaw DJ, Marsh PD, Allison C, Schilling KM. Eff ect of associated dental plaques. Microbiology 2007;153(Pt Engl J Med 2005;353(18):1899-911. oxygen, inoculum composition and fl ow rate on development 11):3809-16. 44. Paster BJ, Boches SK, Galvin JL, et al. Bacterial diversity in of mixed-culture oral biofi lms. Microbiology 1996;142 ( Pt 82. Porter J, Edwards C, Morgan JA, Pickup RW. Rapid, human subgingival plaque. J Bacteriol 2001;183:3770-83. 3):623-9. automated separation of specifi c bacteria from lake water 45. Kazor CE, Mitchell PM, Lee AM, et al. Diversity of bacterial 64. Marsh PD. Dental plaque: biological signifi cance of a biofi lm and sewage by fl ow cytometry and cell sorting. Appl Environ populations on the tongue dorsa of patients with halitosis and and community life-style. J Clin Periodontol 2005;32 Suppl Microbiol 1993;59(10):3327-33. healthy patients. J Clin Microbiol 2003;41(2):558-63. 6:7-15. 83. Beall CJ, Campbell AG, Dayeh DM, et al. Single cell 46. Bik EM, Long CD, Armitage GC, et al. Bacterial 65. Whitehead NA, Barnard AM, Slater H, Simpson NJ, genomics of uncultured, health-associated Tannerella BU063 diversity in the oral cavity of 10 healthy individuals. ISME J Salmond GP. Quorum-sensing in Gram-negative bacteria. FEMS (Oral Taxon 286) and comparison to the closely related 2010;4(8):962-74. Microbiol Rev 2001;25(4):365-404. pathogen Tannerella forsythia. PLoS One 2014;9(2):e89398. 47. Ouverney CC, Armitage GC, Relman DA. Single- 66. Sturme MH, Kleerebezem M, Nakayama J, et al. Cell to 84. Datta AR, Moore MA, Wentz BA, Lane J. Identifi cation cell enumeration of an uncultivated TM7 subgroup in cell communication by autoinducing peptides in gram-positive and enumeration of Listeria monocytogenes by nonradioactive the human subgingival crevice. Appl Environ Microbiol bacteria. Antonie Van Leeuwenhoek 2002;81(1-4):233-43. DNA probe colony hybridization. Appl Environ Microbiol 2003;69(10):6294-8. 67. Bassler BL, Losick R. Bacterially speaking. Cell 1993;59(1):144-9. 48. Kumar PS, Griff en AL, Barton JA, et al. New bacterial 2006;125(2):237-46. 85. Vartoukian SR, Palmer RM, Wade WG. Cultivation of a species associated with chronic periodontitis. J Dent Res 68. Nichols D, Lewis K, Orjala J, et al. Short peptide induces Synergistetes strain representing a previously uncultivated lineage. 2003;82(5):338-44. an “uncultivable” microorganism to grow in vitro. Appl Environ Environ Microbiol 2010;12(4):916-28. 49. Colombo AP, Boches SK, Cotton SL, et al. Comparisons of Microbiol 2008;74(15):4889-97. 86. Vartoukian SR, Downes J, Palmer RM, Wade WG. subgingival microbial profi les of refractory periodontitis, severe 69. Mukamolova GV, Kaprelyants AS, Young DI, Young M, Fretibacterium fastidiosum gen. nov., sp. nov., isolated from periodontitis and periodontal health using the human oral microbe Kell DB. A bacterial cytokine. Proc Natl Acad Sci U S A the human oral cavity. Int J Syst Evol Microbiol 2013;63(Pt identifi cation microarray. J Periodontol 2009;80(9):1421-32. 1998;95(15):8916-21. 2):458-63. 50. Hugenholtz P. Exploring prokaryotic diversity in the genomic 70. Cohen-Gonsaud M, Barthe P, Bagneris C, et al. The structure 87. Kistler JO, Pesaro M, Wade WG. Development and era. Genome Biol 2002;3(2):REVIEWS0003. of a resuscitation-promoting factor domain from Mycobacterium pyrosequencing analysis of an in-vitro oral biofi lm model. BMC 51. Ferrari BC, Binnerup SJ, Gillings M. Microcolony tuberculosis shows homology to lysozymes. Nat Struct Mol Biol Microbiol 2015;15:24. cultivation on a soil substrate membrane system selects for 2005;12(3):270-3. 88. Thompson H, Rybalka A, Moazzez R, Dewhirst FE, Wade previously uncultured soil bacteria. Appl Environ Microbiol 71. Keep NH, Ward JM, Cohen-Gonsaud M, Henderson B. WG. In vitro culture of previously uncultured oral bacterial 2005;71(12):8714-20. Wake up! Peptidoglycan lysis and bacterial nongrowth states. phylotypes. Appl Environ Microbiol 2015;81(24):8307-14.

JULY 2016 455 oral bacteria

CDA JOURNAL, VOL 44, Nº7

89. Jung D, Seo EY, Epstein SS, et al. Application of a new cultivation technology, I-tip, for studying microbial diversity in freshwater sponges of Lake Baikal, Russia. FEMS Microbiol Ecol 2014;90(2):417-23. 90. Gavrish E, Bollmann A, Epstein S, Lewis K. A trap for in situ cultivation of fi lamentous actinobacteria. J Microbiol Methods 2008;72(3):257-62. 91. Chan KG, Yin WF, Sam CK, Koh CL. A novel medium for the isolation of N-acylhomoserine lactone-degrading bacteria. J Ind Microbiol Biotechnol 2009;36(2):247-51. Paul Maimone 92. Tamaki H, Hanada S, Sekiguchi Y, Tanaka Y, Kamagata Y. Broker/Owner It is a Great Time to Sell! Inventory & Eff ect of gelling agent on colony formation in solid cultivation

Rates are Still Low & Buyer Demand is of microbial community in lake sediment. Environ Microbiol 2009;11(7):1827-34. High! Call for a Free in Office Valuation! 93. Aoyagi H, Kuroda A. Eff ects of low-shear modeled microgravity on a microbial community fi ltered through a 0.2- ARCADIA – (4) op comput G.P. Located in a well known Prof. Bldg. on a main thoroughfare. mum fi lter and its potential application in screening for novel Cash/Ins/PPO pt base. Annual Gross Collect $300K+ on a (3) day week. REDUCED microorganisms. J Biosci Bioeng 2012;114(1):73-9. BAKERSFIELD #31 - Free Stand. Bldg. & Pract. (4) op comput G.P. w excell. exposure & 94. Stott MB, Crowe MA, Mountain BW, et al. Isolation of novel th signage. (3) ops eqt./4 plumbed. Annual Gross Collect $325K+ Cash/Ins/PPO. bacteria, including a candidate division, from geothermal soils in CLAIREMONT – (3) op comput G.P. w newer eqt. 2015 Collect $265K Cash/PPO. SOLD New Zealand. Environ Microbiol 2008;10(8):2030-41. GROVER BEACH - (3) op Turnkey Office w included charts (not guaranteed). (2) ops eqt’d w 95. Bollmann A, Palumbo AV, Lewis K, Epstein SS. Isolation and newer eqt. 3rd plmbed. Digital Pano & x-ray. Dentrix. In a strip ctr. LL incentives. PENDING MONTEBELLO - (4) op comput G.P. (2) ops eqt’d. Located in a busy shop. ctr. w exposure & physiology of bacteria from contaminated subsurface sediments. visibility. Annual Gross Collect. $200K on a p.t. schedule. Cash/Ins/PPO. Seller retiring. Appl Environ Microbiol 2010;76(22):7413-9. MONTEREY PARK – (6) op comput G.P. located in a street front suite on a main thoroughfare 96. Aoi Y, Kinoshita T, Hata T, et al. Hollow-fi ber membrane w exposure/visibility. Cash/Ins/PPO/Small % Denti-Cal. Gross Collect $250K+ p.t. REDUCED chamber as a device for in situ environmental cultivation. Appl OXNARD #9 - (3) op comput G.P. & a Prof Office Condo for sale. Located on a main Environ Microbiol 2009;75(11):3826-33. thoroughfare. (3) ops eqt’d. Annual Gross Collect $200K+ p.t. Cash/Ins/PPO/HMO $4.5K/mos 97. Ben-Dov E, Kramarsky-Winter E, Kushmaro A. An in situ method Cap Cks. Digital x-rays. Low overhead. Buy & Combine or open a satellite. NEW for cultivating microorganisms using a double encapsulation SANTA BARBARA COUNTY – (3) op comput G.P. & a 1,900 sq ft Bldg. that houses the technique. FEMS Microbiol Ecol 2009;68(3):363-71. practice & a residential unit that can be rented or lived in. “Fee for Service.” No PPO, HMO or 98. Park J, Kerner A, Burns MA, Lin XN. Microdroplet-enabled Denti-Cal. 2015 Gross Collections ~ $275K on a relaxed 3½ day week. Seller refers all O.S., highly parallel co-cultivation of microbial communities. PLoS One Perio, Ortho, Endo & implant placement. Seller retiring but will assist w transition. 2011;6(2):e17019. So. EAST KERN COUNTY - (5) op comput. G.P. located in a free stand bldg. w exposure/ 99. Moon J, Kim J. Isolation of Paenibacillus pinesoli sp. nov. from visibility & signage. VERY LIMITED COMPETITION. 2015 Collect $600K. Cash/Ins/PPO. Digital x-rays & CT Scan. (6) sensors, Bldg. also available. Seller retiring. NEW forest soil in Gyeonggi-Do, Korea. J Microbiol 2014;52(4):273-7. SAN FERNANDO VALLEY #9 - (8) op comput. G.P. w modern eqt. In a prof. bldg. on a main 100. Tanaka Y, Benno Y. Application of a single-colony co-culture thoroughfare. Cash/Ins/PPO/HMO. Cap Ck approx $7K/mos. 2015 Collect $1.4M+ PENDING technique to the isolation of hitherto unculturable gut bacteria. SAN FERNANDO VALLEY #10 - Located in an exclusive area of the Valley. (5) op comput Microbiol Immunol 2015;59(2):63-70. G.P. w high end buildout. Digital x-ray and CT Scan, Laser, Dentrix s/w & (5) year old eqt. Gross 101. Vartoukian SR, Palmer RM, Wade WG. Cultivation of a Collect $1M+/yr. Cash/Ins/PPO pts. Reasonable overhead, high Net! NEW Synergistetes strain representing a previously uncultivated lineage. SAN GABRIEL VALLEY - (4) op comput G.P. w newer P&C Chairs/Eqt, All the toys & Env Microbiol 2010;12(4):916-28. whistles. Paperless, Schick digital x-rays, Solaris Steril Ctr, Soprocare Intra Oral Camera, 102. Thompson H, Rybalka A, Moazzez R, Dewhirst F, Wade W. Velscope Cancer Screen, The Wand, Air Abrasion, Electric Hand Pieces, Laser, etc. FFS, 2015 In-vitro culture of previously uncultured oral bacterial phylotypes. Gross Collect. $881K+ on a 3½ day week. (4) days of Hygiene. Seller retiring. NEW Appl Env Microbiol 2015;Sept 25, Epub ahead of print. SANTA ANA - absentee owned (6) op fully eqt’d G.P. First floor street front location on a main 103. Morris JJ, Johnson ZI, Szul MJ, Keller M, Zinser ER. thoroughfare. Exposure/visibility/signage. Cash/Ins/PPO. No HMO & No Denti-Cal. Pano eqt’d & Comput. Annual Gross Collect. ~ $500K on a (3½) to (4) day week. BACK ON MARKET Dependence of the cyanobacterium Prochlorococcus on THOUSAND OAKS (4) ops/(2) eqt’d comput. Turnkey Office w included charts. Chart included hydrogen peroxide scavenging microbes for growth at the but not guaranteed. Sirona Eqt. Located in a condo in a Prof. Bldg. on a main thoroughfare. ocean’s surface. PLoS One 2011;6(2):e16805. UPCOMING PRACTICES: Bakersfield, Beverly Hills, Central Coast, Covina, Downey, 104. Nichols D, Cahoon N, Trakhtenberg EM, et al. Use of ichip Duarte, Goleta, Oxnard, Pomona, San Gabriel, Palm Desert, Van Nuys, Visalia & West L.A.. for high-throughput in situ cultivation of “uncultivable” microbial species. Appl Environ Microbiol 2010;76(8):2445-50. D&M SERVICES: Q Practice Sales and Appraisals Q Practice Search & Matching Services THE CORRESPONDING AUTHOR, William Wade, BSc, PhD, can be Q Practice and Equipment Financing Q Locate and Negotiate Dental Lease Space reached at [email protected]. Q Expert Witness Court Testimony Q Medical/Dental Bldg. Sales & Leasing Q Pre - Death and Disability Planning Q Pre - Sale Planning P.O. Box #6681, WOODLAND HILLS, CA. 91365 Toll Free 866.425.1877 Outside So. CA or 818.591.1401 www.dmpractice.com Serving CA Since 1994 CA BRE Broker License # 01172430

CA Representative for the National Association of Practice Brokers (NAPB)

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 RM Matters CDA JOURNAL, VOL 44, Nº7

Preservation of Property: A Critical Obligation TDIC Risk Management Staff

o, a piece of equipment at your of $30,000-$50,000, not including practice has stopped working. loss of income. Most claims were Maybe it was the air compressor. Under most policies, some due to water damage, typically Maybe it was the autoclave. resulting from the failure of a water Maybe it was the vacuum causes are covered, while supply line to dental equipment. Ssystem. Whatever the case, the priority some are not. The burden of A common scenario is this: is to get it up and running as soon as proof to establish that the Unbeknownst to the practice owner, possible. Luckily, you’ve called out a cause of the loss is covered there is a point of weakness in the technician who can replace the broken water system. Perhaps there is a loose equipment right away, leaving a loaner rests with the insured. compression fi tting, a worn valve or a in its stead. He’ll even take the broken tiny hole in a piece of fl exible tubing. At equipment off your hands, saving you night, or over a weekend, when the water the headache of disposing of it yourself. Not so fast. When faced with equipment failure, practice owners must be prudent with their actions should they seek to fi le a claim with their insurance carrier. At best, failure to preserve property can result in a delay in payment; at worst, it can result in the loss not being covered at all. You are not a sales goal. Some practice owners are unaware that many insurance companies, including The Dentists Insurance Company, have the right to inspect malfunctioning equipment in order to determine the cause of failure. Under most policies, some causes are covered, while some are not. The burden of proof to establish that the cause of the loss is covered rests with the insured. You are a dentist deserving of an insurance company relentless “It is a policy requirement that we in its pursuit to keep you protected. At least that’s how we see are provided an opportunity to inspect it at The Dentists Insurance Company, TDIC. Take our Risk the broken equipment, and along with this requirement is the policyholder’s Management program. Be it seminars, online resources or our responsibility to preserve the property in Advice Line, we’re in your corner every day. With TDIC, question,” said Sheila Davis, assistant vice you are not a sales goal or a statistic. You are a dentist. president, Claims and Risk Management Claims, TDIC. “This is essential so that we can determine whether the loss is ® covered. Disposal of property without Protecting dentists. It’s all we do. our go-ahead could affect your claim.” 800.733.0633 | tdicinsurance.com | CA Insurance Lic. #0652783 In 2015, TDIC had a total of 446 property claims with an average value

JULY 2016 459 JULY 2016 RM MATTERS

CDA JOURNAL, VOL 44, Nº7

is not being used, the water pressure builds be used to obtain this information. But independently. If there is no equipment and the dam bursts, fl ooding the offi ce. the reality is, most of these “reports” are to inspect, it is diffi cult, if not impossible, When faced with a situation like just invoices; they often lack the details to hold the at-fault party accountable. this, practice owners need to follow needed to make a determination of cause. In some cases, practice owners don’t certain protocols. Because of the “We need to know exactly how want the equipment taking up precious complex nature of dental equipment, the equipment malfunctioned and offi ce space, nor do they know what to they should preserve not only the entire why it failed. Tech reports don’t do with the broken equipment once a mechanism, but the failed parts as well. generally disclose this,” Davis said. claim is in process. But more often than “Each of these can usually be examined In addition, the opportunity to recover not, technicians will be happy to return to determine why the failure occurred the amounts paid in the claim from the for the equipment in a few days, after the and which part failed,” Davis said. “But responsible party may be lost if the cause insurance representative has had a look. if the equipment is disposed of, then of the damage is disposed of. For example, “For any type of equipment breakdown, the opportunity to determine how and should an insurance carrier determine it’s better to err on the side of caution why the failure occurred is lost.” the loss was caused by a manufacturer’s and keep the equipment. In most cases, Some practice owners erroneously defect, the manufacturer would have we can send out someone to inspect the assume the repair technician’s report can a right to inspect the equipment equipment or failed component the same day or the following day,” Davis said. In one recent case, a dentist experienced the failure of her vacuum. Knowing she couldn’t afford to close her practice during the claims process, she replaced it, storing the broken one onsite. TDIC was able to get an inspector out to her practice right away, and she was able to continue seeing patients while her claim was being processed. “We understand that you can’t afford to have downtime,” Davis said. “But by calling us in tandem with calling a technician, and by preserving your old equipment, you can ensure your claim will be processed smoothly.” Experiencing an equipment breakdown is an unfortunate reality of the dental profession. As a practice owner, the steps you take during this time can mean the difference between a smooth recovery or a complicated one. By following a few simple protocols, you can get back to business quickly and painlessly. ■

TDIC’s Risk Management Advice Line at 800.733.0634 is staffed with trained analysts who can answer property insurance and other questions related to dental practice.

460 JULY 2016 (;3(5,(1&(7+(',))(5(1&( t Lee Skarin and Associates has been serving the dental profession since 1959.

,1& t Kurt Skarin has over 30 years experience in dental practice sales.  t We have sold more practices than any broker in the state within the last 12 months. t Our experienced practice appraisals are backed with credentials unequaled among dental practice brokers. t We provide in-house legal counsel to advise you in all aspects of the sale and purchase, including the tax consequences of the sale. t Excellent financing is available, in most cases for 100% of the purchase price. t With a reputation for experienced, concientious, and ethical performance, we give our clients personal attention in all aspects of the purchase.

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With scores of Buyers, profiles of their practice interests and financial ability, /HH6NDULQ $VVRFLDWHV is able to find the right buyer for your practice. 2IÀFHV

Experience the difference. Call Lee Skarin and Associates  for responses to all of your questions - No obligation!  Visit our website for current listings: www.LeeSkarinandAssociates.com Dental Practice Brokers CA DRE #00863149 800.752.7461 Specialists in the Sale and Appraisal of Dental Practices Practices Serving California Dentists since 1966 How much is your practice worth?? Wanted Selling or Buying, Call PPS today!

NORTHERNNORT CALIFORNIA SOUTHERN CALIFORNIA (415) 899-8580 – (800) 422-2818 (714) 832-0230 – (888) 440-5957 Raymond and Edna Irving Thomas Fitterer and Dean George [email protected] [email protected] www.PPSsellsDDS.com www.PPSDental.com California DRE License 1422122 California DRE License 346937 6109 NORTHRTH LAKE LAKE TAHOE “Best-of-the-best!”“Best-of-the-best!” 6ROLG6R ANTELOPE VALLEY Has grossed $1.8 Million. Fantastic location. foundation anchored by 8.5 days of Hygiene. Consistent $1 60,000 autos pass by per day. 8 ops. Partnership for $250,000 or buy all. MillionSHUyear performer. Beautiful office with unsurpassed ARCADIA Facility only. 3-ops equipped. $65,000 or $95,000 with Ortho. views. BAKERSFIELD AREA 5-ops, next to McDonalds. 1,800 sq.ft. includes 6108 SANTA ROSA 3-day practice collected $320,000.7ZR building. Grosses $40,000/month. Full Price with building $350,000. days of hygiene, 3-ops and 500+ active patients. BAKERSFIELD Established 55 years. 5-ops in 3,000 sq. ft. Will do $1 Million. Full Price $300,000. Building available for $350,000. 6107 (85(.$ 100% out of network with WKHinsurance BELLFLOWER Established 60-years. Grossing $350,000. Full Price industry. Produced $918,000 and collected $895,000 on 20- $240,000. hour week. 7+ days of Hygiene. EAST LOS ANGELES One million Latinos in service area. PPS sold 6106 FOLSOM AREA No rush and no chaos here. Staff is a to Seller in 1985. Will do $1 Million in 18 months. Full Price $300,000. Dream Team. Beautiful facility. 2015 collected $640,000. EAST SAN FERNANDO VALLEY Absentee Owner. $8,000SHU Unique opportunity seeks Dentist looking for something special. month Cap Check. 4-ops. Do a Million within a year. 6105 MODESTO  Collected $430,000+ on 3-day week. 3- INDIO 4,000 sq.ft. dental building. Full Price $650,000. days of Hygiene. 5-ops. Central location. Successor should LADERA RANCH Grossing $650,000. Shopping center location. open 4th day. LAGUNA NIGUEL Location, location, location! 4-ops with Panorex. 6104 SANTA CLARA – CUPERTINO AREA Restorative Full Price $185,000. practice. 2015 collected $1.55 Million with Profits of $650,000. LA JOLLA Established 20-years. 3-ops. Grossed $150,000. Super Paperless. Beautiful office. Extremely attractive selling features opportunity with immediate growth. Full Price $150,000. to retain goodwill. LAWNDALE Hi identity. 2 ops . Full price $125,000. 6103 SAN FRANCISCO’S UNION SQUARE Opportunity to LOS ANGELES HMO Grossing $1.2 Million. 5-ops. Full Price $1.2 Million. acquire highly regarded practice with condo. Beautiful 5-ops, LOS ANGELES HMO Does $4 Million. SOLD digital and paperless. 6th op available. 2015 collected $658,000. NORCO – CORONA Will do $1.5 Million. 8-ops. Exquisite. Full Price $1.2 Million. 6102 SAN RAFAEL 2-ops. 3-day week, 2015 collected NORWALK Fantastic high identity location. 5 ops. Full Price $259,000. 2014 saw $332,000. Full price $150,000. $250,000. SOLD 6100 SANTA CLARA Phenomenal launching pad for Ambitious ORAL SURGERY PRACTICE – LOS ANGELES Established 40 years. Successor. Fantastic location, 5-op facility. Management not ORANGE Beautiful 10 operatory office ready for merger. taking advantage of what is possible even though 2015 collected PASADENA Established 60 years. 7-ops. Always $1+ Million. Full $758,000 with Profits of $323,000. Positioned to be $1 Million+ Price $600,000. year performer immediately! REDLANDS Shopping center. Grosses $350,000. Full Price $250,000. 6099 FAIRFIELD Collected $500,000 in 2015. 3-days of RIVERSIDE Facility only. 4 ops. Full Price $50,000. Hygiene. 4-ops with digitalSOLD radiography. SOUTH ORANGE COUNTY BEACH CITY Grosses $650,000. 6098 WEST PETALUMA Petaluma is QRZthe hip business 4 ops. Beautiful! center RI the North Bay! Collected $468,000 with Profits of PERIO PRACTICE - PRESTIGIOUS BEACH CITY Established $199,000 3-days of Hygiene with 4th GD\starting SeptHPEHU. 40 years. 6096 NORTH FRESNO’S ST. AGNES MEDICAL VILLAGE TORRANCE Established 12 years. 5 star building. 3-ops. Grossing 4-days of hygiene. Collected $425,000 in 2015. 4-ops with 3 $250,000. Full Price $195,000. upgraded. Full Price $150,000.SOLD TUSTIN Dental building. Full Price $1.5 Million. 6094 PERIO PRACTICE - SAN FRANCISCO BAY AREA VENTURA - OXNARD 5-ops. Grossing $850,000. High identity. Full Price $685,000. Shall appeal to Perio desiring high-end practice in very desirable area. 2015 Produced $1.25 Million, collected $1.25 Million and YUCCA VALLEY 8/10th of an acre. Great highway visibility. Full Price $250,000. realized Profits of $690,000 on 3-day week. 6089 MOUNT SHASTA Small town living renowned for Seeking Senior Dentists wishing to have more time to enjoy life, be free outdoor lifestyle. Escape from Rat Race and corporate intrusion. of management & overhead to join a Dental Cooperative. Call Tom 3-day week collected $900,000. Strong Profits. Fitterer at 714-832-0230 or cell 714-345-9659. 6070 VISALIA 7KLVSUDFWLFHLVZHOOSRVLWLRQHGIRULWVQH[W **FOUNDERS OF PRACTICE SALES** FDUHWDNHUStrong Hygiene Department, beautiful facility, well 120+ years of combined expertise and experience! equipped. Digital throughout. Collected $727,000 on part-time 3,000+ Sales - - 10,000+ Appraisals scheduleLQ. Extend hours and be busier. Best location! **CONFIDENTIAL** PPS Representatives do not give our business name when returning your calls. Regulatory Compliance CDA JOURNAL, VOL 44, Nº7

A Patient’s Right to Access Records Q-and-A CDA Practice Support Staff

he U.S. Department of Health a request be written and that its own What is considered to be the patient’s and Human Services (HHS) form be used. The requirement to record? released in March 2016 a use a written access request must HIPAA gives a patient the right guidance document and FAQ be noted in the covered entity’s to review or obtain a copy of his or on the Health Insurance Notice of Privacy Practices. A her information maintained in a TPortability and Accountability Act of covered entity may offer electronic covered entity’s “designated record 1996 (HIPAA) requirements for options for making the request (for set.” The designated record set is that providing a patient with access to his or example, a web portal or email), but group of records maintained by or her record. This article highlights some it cannot require the use of those for a covered entity that is used, in of the key points in the guidance, which options. Any requirement to use a whole or part, to make decisions about is found at hhs.gov/hipaa. More Q-and- covered entity’s form may not create an individual, or that is an entity’s As are included in the CDA Practice a barrier or unreasonably delay a billing and payment records for that Support resource, Patient Request to patient from obtaining access. individual. The designated record set Access Records (Records Release) Form and Q-and-As, which are available at cda.org/practicesupport. HHS provided new information on the following topics: When Looking To Invest In Professional ■ Fees, particularly limits on what can be charged. Dental Space Dental Professionals Choose ■ Avoidance of unreasonable delay to patient. ■ Access in the form or format of the patient’s choosing, including unencrypted email. Linda Brown

What does “right to access record” mean? 30 Years of Experience Serving It means a health care provider must: the Dental Community Proven ■ Allow a patient to inspect his or her record. Record of Performance ■ Provide a copy or summary of the record if requested by the • Dental Office Leasing and Sales patient. ■ Transmit a copy of the record to For your next move, • Investment Properties a person or entity of the patient’s contact: LINDA BROWN • Owner/User Properties choosing. Requests for this type of access must be written. • Locations Throughout Direct: (818) 466-0221 Southern California Must the access request be in writing? Office: (818) 593-3800 State law requires health care Email: [email protected] providers to comply with written Web: www.TOLD.com requests for access, but does not Cal BRE: 01465757 expressly require only written requests. A HIPAA-covered entity may require

JULY 2016 463 JULY 2016 REGULATORY COMPLIANCE

CDA JOURNAL, VOL 44, Nº7

may include information generated labor, supplies and postage. from the same address as the by other health care providers that is The fee for providing a one collected from the patient maintained by the covered entity. summary must be agreed to by at the fi rst appointment. The record includes images, the patient in advance. ■ Signature and information impressions and models if they If a patient requires a copy of a on a written request matches have been used to make decisions portion of his or her record to support that in the record. about an individual’s treatment. an appeal regarding eligibility for a public benefi t program, such as Denti- The patient is requesting an electronic What may I charge? Cal, the copy shall be provided by the copy, but I keep paper records. Am I The HHS guidance made clear that dental offi ce at no charge. The patient required to provide an electronic copy? the fee for access may include only the is entitled to no more than one copy If the dental practice is a HIPAA- cost of: free of charge, but may not be limited covered entity, the answer is yes. ■ Labor to make the requested in the number of requests for copies. In its March 2016 guidance, HHS copy, whether in paper Dental practices that are clarifi ed several issues related to the or electronic form. not HIPAA-covered entities form and format of copies. Generally ■ Supplies such as paper or must follow the state’s rules and speaking, a covered entity must portable electronic media. may charge no more than: comply with a patient’s request for ■ Postage when the patient requests ■ Twenty-fi ve cents per page for a specifi c form and format unless it the copy or summary be mailed. copying paper documents. is not readily producible. Examples ■ Preparation of an explanation ■ Fifty cents per page of form and format are paper, fi lm, or summary of the record if from microfi lm. electronic/pdf, electronic/jpg and requested by the patient. ■ Actual cost for duplicating electronic/DICOM or .dcm. A covered entity may either X-rays, photos, models, If the form and format requested by calculate actual labor costs to impressions, etc. the patient is not readily producible fulfi ll a request or develop a fee ■ Actual postage cost. by the covered entity, both parties schedule based on average labor In addition, such a dental should agree on an acceptable format. costs to fulfi ll a request. practice may charge a fee based on A dental practice that is not a The fee may not include costs reasonable clerical costs incurred HIPAA-covered entity is not required associated with verifi cation of the in locating and making the records to provide electronic copies. request, documentation, searching for available for inspection. and retrieving the record, maintaining We always use a secure method to systems, recouping capital for data What are acceptable methods of send patient information electronically. access, storage or infrastructure, or verifying the access request from a A patient is requesting we send his anything not included in the above patient or patient’s representative? information to him via unencrypted paragraph. A per-page fee may not All dental practices must take email. What do we need to do to be charged for records maintained reasonable steps to verify the identity comply with the patient’s request? electronically. If a dental practice of the person making the request A dental practice must (1) collects fees, it should prepare a for access. There is no one required advise the patient of the risks of document listing the fees and provide method of verifi cation. A patient unsecure electronic transmission of it to the patient with the Patient may not be required to be present information and (2) the patient must Request to Access Records form. to make an access request. Methods consent to the unsecure electronic A covered entity may charge of verifying identity include: transmission of information before a flat fee for standard requests for ■ Checking identifi cation the dental practice can send the electronic copies of electronic of the individual making information via unencrypted email. records, provided the fee does the request in person. not exceed $6.50, inclusive of all ■ The emailed request was sent CONTINUES ON 466

464 JULY 2016

“Matching the Right Dentist CARROLL to the Right Practice” &COMPANY

CComplete Evaluation of Dental Practices & All Aspects of Buying and Selling Transactions

4103 SAN FRANCISCO GP 4110 SANTA ROSA GP Vibrant downtown location in historic high-rise bldg. Retiring doctor Don’t miss this opportunity ~ absolutely gorgeous, state of the art offering 30+ years of goodwill. 4.5 days of hygiene, 1,500+ active office located within two major thoroughfares in the heart of Santa patients (all fee-for-service). Beautiful, spacious shared facility in Rosa. Practice generating $2.1M+ in GR on 5 dr. days/week with 9 approx. 2,500 sq. ft. Seller has use of 4 ops (3 fully equipped and 1 days of hygiene. 2015 Adj net over $569K. 3,000+ active patients, overflow op). 2015 GR $796K. 2014 GR $768. Average adjusted 9 fully equipped ops in spacious 2,950 sq. ft. Equipment includes net income $274K+ Asking $599K. digital x-rays, Sirona-D Cone Beam system, Cerec Blucam and Ominicam. Asking $1,436,000. 4085 SANTA ROSA GP & BUILDING Practice and real estate are offered for sale in a well-established 4093 SAN JOAQUIN VALLEY ORTHO condominiumized medical/dental complex conveniently located in Established over 35 years with a solid reputation, near several referral the heart of Santa Rosa, near Memorial Hospital. This 1,200 sq. ft. sources in seller owned building. 2,500 sq. ft. office with 7 chair single story office comes equipped, furnished and ready for you to open bay in professional center on a well-travelled street with many continue your professional career with established and new patients retailers. Avg. Gross Receipts $763K. Seller retiring and willing to (approx. 750 active patients). Seller is retiring after almost 20 years help for smooth transition. Asking $561K. The building is available to but will assist for a smooth transition. Average Gross Receipts of purchase as well for $608K. $256K with adj. net of approx. $110K. Asking price $160K for the practice, and $270K for the real estate. 4086 SILICON VALLEY PERIO Well-established Perio practice in prime San Jose location with 4108 HUMBOLDT COUNTY GP referral sources nearby. Located in a commercial & residential mix Well-established, high performing general practice boasts 6 fully neighborhood with a large daytime business draw. Approx. 1,100 equipped ops. in 2,900 sq. ft. free standing office w/Digital X- ray, 2 sq. ft. office with 4 fully-equipped ops. Well trained dedicated staff, platinum Dexis sensors, & Cerec Omnicam & MCXL units. Loyal & seller retiring and willing to help for smooth transition. 2014 GR stable pt. base in charming community, w/ a small town feel. Perfect $482K+, 2015 on schedule for $539K+ as of August. Asking for a dentist who wants to escape the grind and live along the $295K. coastline. Avg. GR $1.4M+, 2016 on schedule for $1.5M+. Seller willing to help for smooth transition. Asking $1,041,000. 4065 LAKE COUNTY GP Seller retiring from general practice located in a slower paced, 4091 HOLLISTER GP & PEDIATRIC relaxed community. Plenty of hunting and fishing and out door Country living at its best ~ small town community feel with affordable activities for the enthusiast. Approximately, 1,600 square foot office housing, in quaint bedroom community to Silicon Valley. General and with 4 fully-equipped operatories. Over 2,000 active patients, Pediatric practice located in corner professional building on well- average $697K+ in Gross Receipts with an overhead of just 56%, travelled street near Hazel Hawkins Hospital. Fully-equipped 1,600 and 4 doctor days per week. Asking $463K. sq. ft. office with 2 enclosed adult ops and 3 open pedo ops. Great opportunity for a turn key practice with trained staff and 4105 STANISLAUS COUNTY GP approximately 700 active patients. 2014 GR $228K. Seller is The Bay Area is getting over crowded. Get away to a less relocating out of the area, but will help for a smooth transition. Asking demanding commuter friendly town. Seller retiring from practice est. price only $125K. over 30 years ago with loyal patient base in charming community with historic small town feel. 3 fully-equipped ops. in 1,200 sq. ft. 4114 CONCORD GP office. Approx. 1,400 active pts. w/4 doctor days/week. 5 year avg. Retiring owner offering a well-established, Concord practice, in GR $647K+ w/approx. 50% overhead. Seller willing to help for desirable community with 30+ years of goodwill. Strategic location smooth transition. Asking $428K. with growth potential. 3 fully-equipped ops. in 836 sq. ft. facility. 5 year Avg. GR $360K+ with 2 doctor days. Owner available for a UPCOMING: smooth transition. Asking $224K. 4116 SACRAMENTO COUNTY ORTHO 4115 WALNUT CREEK GP Walnut Creek practice in gorgeous facility with recent leasehold Carroll & Company improvements plus new and upgraded equipment. Practice has 30+ 2055 Woodside Road, Ste 160 years of goodwill. Looking for a mature, experienced practitioner for a Redwood City, CA 94061 loyal and mature patient base. Located in commercial center with P (650) 362-7004 several amenities and marketing opportunities. Doctor works 2 days F (650) 362-7007 per week. Owner available for a smooth transition. Asking $432K. [email protected] 4096 MENDOCINO COUNTY GP www.carrollandco.info Seller offering well est. 48 year practice. Located in outdoorsman's BRE #00777682 paradise. Just 2 hours North of SF surrounded by redwood forest, vineyards and mountains. 950 sq. ft. office in single level building w/ Mike Carroll Pamela Carroll-Gardiner 4 fully equipped ops. 2014 GR $565. Asking $300K.

www.carrollandco.info [email protected] P (650) 362-7004 F (650) 362-7007 JULY 2016 REGULATORY COMPLIANCE

CDA JOURNAL, VOL 44, Nº7

CONTINUED FROM 464 I want to transmit a patient’s entity is not liable for what Situations for which a dental practice information to a specialty dentist via happens to the PHI once the may want to use the consent form are: unencrypted email — do I need to get designated third party receives ■ To obtain an adult child’s the patient’s authorization to do so? the information as directed by the consent to share detailed HIPAA allows a covered entity individual in the access request.” information as many times as to share patient information with needed during a time period with another covered entity without the The patient requests that I mail the the parents who are the payers. patient’s authorization if the purpose of copy to an individual. May I ask the ■ To market products or sharing the information is the patient’s patient to pick up the copy instead? services to a patient. treatment. HIPAA requires this No, you may not. Such a ■ To participate in research. ■ information sharing be done securely, request may be viewed as a so a patient’s authorization to share barrier to the patient’s right to Regulatory Compliance appears the information via unsecure methods access his or her record. monthly and features resources about is insuffi cient to waive the covered laws and regulations that impact dental entity’s obligation. To be able to send A new patient has requested a copy practices. Visit cda.org/practicesupport it via unencrypted email requires the of his records from his former dentist for more than 600 practice support patient make an access request and but the dentist is refusing to provide resources, including practice management, direct you to send the information to them. What can the patient do? employment practices, dental benefi t the specialist via unencrypted email. Suggest that the patient submit to plans and regulatory compliance. the other practice a written request for The patient is requesting an electronic records plus a copy of the CDA Oral copy be sent to her new dentist via Health Fact Sheet on Patient Records unencrypted email — may I do that? or, if it is not a California practice, If the dental practice is a HIPAA- the HHS March 2016 guideline. If covered entity, the answer is yes. the other practice does not comply HHS, in the March 2016 guidance, with the request, the patient can fi le states the patient’s right to receive a written complaint with the dental information via unsecured electronic board and with the Department communication extends to sending of Health and Human Services. it to a third party at the patient’s request. HHS further states: Who else may have a patient’s “… if the individual requested information, and under what that the covered entity transmit circumstances? the PHI in an unsecure manner Review the resource Uses and (e.g., unencrypted), and, after Disclosures of Patient Health Information being warned of the security found on cda.org/practicesupport. risks to the PHI associated with Requests from others for patient the unsecure transmission, information for purposes not permitted maintained her preference without patient authorization by HIPAA to have the PHI sent in that or California Confi dentiality of Medical manner, the covered entity is Information Act (CMIA) (California not responsible for a disclosure Civil Code section 56 et seq.) must of PHI while in transmission be submitted on a valid authorization to the designated third party, form that meets CMIA and HIPAA including any breach notifi cation requirements. A Consent Form for Use obligations that would otherwise and Disclosure of Patient Health Information be required. Further, a covered is available on cda.org/practicesupport.

466 JULY 2016 Ethics CDA JOURNAL, VOL 44, Nº7

Undertreatment, an Ethical Issue Henrik Hansen, DDS

ost dentists may It is a diffi cult and uncomfortable not think that situation for a subsequent treating undertreatment can be Involve patients in the decision- dentist to inform a patient that there an ethical issue, but it making process by fully are problems that should have been can be very signifi cant. informing them in language addressed, but weren’t. This can be MLet me relay a true story that was told a very tricky discussion to have with to me in dental school. A dentist was that they can clearly a patient. Dentists have an ethical doing an exam for his mother when he understand, thus ensuring the obligation to be honest, but not to noticed a small lesion on her tongue. ethical principle of autonomy. infl ame the patient by speculation or It didn’t look too awful and he knew denigrating the previous dentist. It if he sent her for a biopsy, it would be is also good practice to contact the painful. The dentist decided to watch previous dentist and have an honest it, and watch it he did. Each time he discussion regarding your fi ndings to saw the lesion, it got a little bit bigger, more diffi cult. We see patients every provide him or her the opportunity to but not too bad in his eyes. However, by day with old restorations that appear respond, as you don’t know all the facts. the time he decided enough was enough serviceable and are asymptomatic. We have a legal and ethical duty to and sent her to an oral surgeon, the However, we know that some of the our patients to diagnose and treatment lesion was a full-blown carcinoma, and restorations will have fractures and/or plan putting their best interests fi rst, his mother ended up with a signifi cant decay underneath that can lead to severe using our best professional judgment portion of her tongue removed. Luckily, consequences if not removed. On the and presenting the information in a it had not metastasized, but his mother other hand, removing the restoration clear and forthright manner. Patients was left with signifi cant morbidity and may make an asymptomatic tooth “hot,” expect and deserve no less. ■ her quality of life was diminished. leading to further complications. What Having just completed six years on should you do? The most reasonable Dr. Hansen is currently a member of CDA’s Council on Peer Review, I have approach is to do a thorough exam the CDA Judicial Council, a California analyzed hundreds of cases from single and inform the patient of your best Delegate to the ADA House of Delegates crowns to full-mouth rehabilitation. clinical judgment. Involve patients in and Vice Regent representing California It is common knowledge that dentists the decision-making process by fully for the International College of Dentists. diagnose and treatment plan differently informing them in language that they Dr. Hansen previously served as chair of based on their training, experience can clearly understand, thus ensuring the CDA Council on Peer Review, was and philosophy, and this is as it should the ethical principle of autonomy. a trustee member of the CDA Board of be. However, Peer Review also sees The cause for overtreatment is Trustees, is a former member of the ADA treatment plans that just don’t make usually economic, but undertreatment Council on Dental Benefi t Programs and sense and are outside the normal range. can be more complex. With the clarity is the Immediate Past Chairman of the The most common undertreatment of hindsight, I can see that when I American College of Dentists, Northern complaints are undiagnosed graduated from dental school 38 years California Section. Dr. Hansen received periodontal disease and decay. ago, I thought I was being conservative his dental degree from the University of Referring to the CDA Code of in my treatment planning; but the California, San Francisco, School of Ethics, it’s clear that under treatment reality was that I had a fear of failure, Dentistry and maintains a private practice in violates the ethical principles of and the less you do, the less you fail. Fairfi eld, Calif. veracity (being honest and telling the Other examples of undertreatment truth), benefi cence (doing good for include not adequately informing For further guidance, contact your local our patients) and integrity (behaving the patient about the importance of ethics committee or Britney Ryan, CDA with honor and decency). When radiographs when they refuse. Being judicial council manager, at 800.232.7645. we look at the ethical principle of too rushed, chemically, emotionally or nonmalefi cence (minimizing harm and age impaired could result in a less-than- maximizing benefi t), things can become acceptable exam and treatment plan.

JULY 2016 467 DENTAL PRACTICE BROKERAGE Making your transition a reality.

Dr. Lee Dr. Thomas Dr. Dennis Dr. Russell Jim Kerri Mario Jaci Steve Thinh Maddox Wagner Hoover Okihara Engel McCullough Molina Hardison Caudill Tran LIC #01801165 LIC #01418359 LIC #0123804 LIC #01886221 LIC #01898522 LIC #01382259 LIC #01423762 LIC #01927713 LIC #00411157 LIC #01863784 (949) 675-5578 (916) 812-3255 (209) 605-9039 (619) 694-7077 (925) 330-2207 (949) 566-3056 (949) 675-5578 (949) 675-5578 (951) 314-5542 (949) 675-5578 25 Years in Business 40 Years in Business 36 Years in Business 33 Years in Business 42 Years in Business 35 Years in Business 35 Years in Business 26 Years in Business 25 Years in Business 11 Years in Business

PRACTICE SALES • PARTNERSHIPS • MERGERS • VALUATIONS/APPRAISALS • ASSOCIATESHIPS • CONTINUING EDUCATION

NORTHERN CALIFORNIA OROVILLE: 1,000 sq. ft.. Dentrix & BEVERLY HILLS:2SV(DJOH6RIW ORANGE COUNTY: (QGRSUDFWLFH2SV Dentrix software, Digital Pan & X-rays, 'LJLWDO&(5(&/RQJWHUPVWDIIQHZHU 'LJLWDODQG3DSHUOHVV\HDUROGHTXLSPHQW BENICIA: Practice & Building. 1,545 sq. ft., /DVHU,QWUD2UDO*5.&$ HTXLSPHQW*50&$ 4 ops, Open-Dental software, digital X-ray & 6WURQJUHIHUUDOEDVHIRU\HDUV&$ Pan, Laser. 2014 GR $550K. #CA298 OROVILLE:2SVUHFHQWO\UHPRGHOHG %5($1HZ/LVWLQJ2SVHVWDEOLVKHG ORANGE COUNTY: 2SV(VW Great satellite or startup practice. Owner \HDUVSURIHVVLRQDOEXLOGLQJ*5 \HDUV'HQWUL['LJLWDO*5RI. BENICIA: VTIWRI¿FHRSV retiring. #CA288 ..$GM1HW&$ ZLWK.$GM1HW&$ *5.GD\ZHHN6LVWHUSUDFWLFH &$VDOHFRPELQHGRUVHSDUDWH&$ PINOLE:2S(QGRRI¿FHZ'LJLWDO;UD\ %85%$1.1HZ/LVWLQJ*HQHUDO3URVWK PASADENA AREA: 2SV'HQWUL['H[LV 0LFURVFRSHVDQG3%6(QGRLQDSSUR[ ,PSODQWSUDFWLFH2SVHTXLSSHG &(5(&HVWDEOLVKHGIRU\UV&$ CITRUS HEIGHTS: Prosthodontic practice VTIW*5.&$ (DV\'HQWDO'LJLWDO*5.. VWDQGDORQHEXLOGLQJIRUVDOH $GM1HW&$ S. ORANGE COUNTY: Pedo Practice with 2SV*5RI&$ ROSEVILLE: 4 equipped Ops, 1 add’l 2SV\HDUQHZHTXLSPHQWGLJLWDO3DQR SOXPEHGVTIWZ'LJLWDO;UD\ CARSON:2SV3DSHUOHVV(DJOH6RIW *5.ZLWKURRPWRJURZ EL DORADO COUNTY:VTIWZ #CA222 /DVHU'HQWUL[VRIWZDUH*5. 'LJLWDO3DQR$OO(TXLS \UVROG 2SVLQWUDRUDOFDPHUDGLJLWDO[UD\ &$ 3UDFWLFH:RUNVVRIWZDUH*5 *5.&$ SANTA BARBARA: 2SVHVWIRU SACRAMENTO: .&$ VTIW(TXLS CENTRAL ORANGE COUNTY: \HDUVGD\V+\JLHQHZHHNORQJWHUPVWDII VWDWLRQVLQED\DGG¶O3OXPEHG*5 ))6*5RI.&$ *5($7(5 (/ '25$'2 +,//6 1HZ .&$ 2SV3DSHUOHVVGLJLWDOEXV\UHWDLOFHQWHU /LVWLQJ  VT IW RIILFH  2SV 'LJLWDO *5RI.ZLWK.$GM1HW SANTA MARIA: New Listing! 2SV 3DQ'LJLWDO6HQVRUV&$ SACRAMENTO: New Listing! 1,000 sq. &$ LQVTIWVXLWH))6332GD\ZRUN IWRI¿FHZHTXLS2SVDGG¶O3OXPEHG COASTAL ORANGE COUNTY: ZHHNZGD\RIK\JLHQH*5. *UHDWHU 5HGGLQJ:  VT IW Z 2SV 'HQWDO0DWHVRIWZDUH*5 2SV(TXLSSHG'HQWUL['H[LV3URIEOGJ &$ ,QWUD2UDO 'LJLWDO 3DQR /DVHU &$' &$ Z\HDUVRIJRRGZLOO*5RI. &$0'HQWUL[*50&$ $GM1HWRI.&$ SOUTH PASADENA: 2SV(TXLSSHG SAN FRANCISCO: Periodontal Practice SDSHUOHVVGLJLWDOHVW\UV*5. GREATER ROSEVILLE/ROCKLIN: 1HZ &RQGR8QLWVTIWZ2S*5 CYPRESS: 2SV\UVRI*RRGZLOO ZLWK.$GM1HW&$ /LVWLQJ  VT IW  2SV ,2 FDPHUD .Z.$GM1HW&$ GD\V+\JLHQHSHUZHHN.*5. VICTORVILLE: 2SV3OXPEHG GLJLWDO [UD\V GLJLWDO 3DQR 0 6FDQQHU SAN JOSE: New Listing! *5. $GM1HW&$ 6T)W(VW\UV6RIW'HQW*5 'HQWUL[VRIWZDUH*5.&$ RQòGD\ZHHN(TXLSRSVDGG¶O )8//(57211HZ/LVWLQJ2SV .&$Price Reduced! GREATER ROSEVILLE/ROCKLIN: 3OXPEHG6T)W(='HQWDO'H[LV (TXLSSHG\HDUVRI*RRGZLOO'HQWUL[ WHITTIER: 2SV(TXLSSHG'HQWUL[   2SV ¿OPEDVHG3DQRUDPLF&$ Z,2 'LJLWDO ;UD\ /DVHU 'HQWUL[ 'LJLWDO3DSHUOHVV*UHDWORFDWLRQ*5 'H[LV(VWIRU\UVRQPDLQVWUHHW RI.&$ VRIWZDUH (VWDEOLVKHG  \HDUV*5 SANTA ROSA: General Dentistry & *5.&$ .&$ %XLOGLQJ2SV*5.Z$GM GREATER LOS ANGELES: Perio SAN DIEGO *5($7(5526(9,//($8%8511HZ 1HW.&$ 3UDFWLFH2SV

A look into the latest dental and general technology on the market

Smoke Free (David Crane, Free) Consumers Expect Small Businesses Dental professionals are considered the fi rst line of defense to Have a Website against oral cancers that may be directly caused by tobacco A website is the No. 1 expectation from consumers when they use. Patient education and help with establishing attainable choose small businesses, more so than on-air advertising, loyalty goals have been among the most eff ective means to help kick programs or a Facebook page. This according to a study of 800 the habit. Smoke Free is an app with the ultimate purpose of consumers conducted by Time Warner Cable titled “Small Business helping smokers quit by providing relevant and useful feedback. Technology Impact Study.” The study found that 36 percent of After entering customized information, which includes how much consumers may not shop at a business that doesn’t have a website. a cigarette pack costs, how many cigarettes are in a pack, how On top of this, 70 percent of the respondents said that having the many cigarettes are smoked per day and how long after waking owners’ photo with a story about the business on the website is up is the fi rst smoke in the morning, smokers can set a target eff ective. These can serve as particularly useful statistics for dentists quit date. After the quit date, the app displays a dashboard who are considering launching a website and a social media of motivational statistics such as the amount of money saved, presence. If dentists were to follow the lead of this study, they number of minutes smoke free and how many cigarettes not would build their website out before fi ring up their Facebook page. smoked. The dashboard statistics rotate to also display money saved over a year, number of minutes of life regained and the — Blake Ellington, Tech Trends editor number of cravings resisted. The app also contains a diary and a series of progress panels that show positive and interactive information to help smokers get through the most diffi cult times to Chrome Browsing Outpacing Safari accomplish their goal. For example, users can create a shopping list and purchase items with the money saved by not smoking. Browsing Users can tap on a button from the dashboard to immediately get Something is changing in the world of mobile and desktop encouraging help when their cravings arise. An in-app upgrade browsing. According to a recent Adobe Digital Index (ADI) study, to the “Pro” version is available, which enables features such as the Chrome browser is gaining more traction with users than the dashboard and progress panel customizations as well as daily iOS-based browser, Safari. Apple users who traditionally have missions to help smokers further their chances of successfully stuck to Safari for their web browsing have to download the quitting. Users can share their progress with anyone directly Chrome browser, which is a sign that it is becoming the preferred from the app. Notifi cations can be enabled for journal and option. Adobe Analytics show that smartphone use of Chrome mission time reminders, in addition to awarding badges for major increased 75 percent over the last year while Safari only saw accomplishments. Simple to use and packed with features, Smoke gains of 33 percent. While the study showed that those who Free is an excellent resource for smokers who are serious in the have an iPhone or iPad still lean toward using Safari, the gap is fi ght to end their addictions. Complete with a plethora of personal shrinking between the iOS-based browser and Google-based positive information and motivational interactive activities to Chrome browser. keep users engaged in their battle to quit smoking, this app is a — Blake Ellington, Tech Trends editor powerful tool that dental professionals can direct their patients to. — Hubert Chan, DDS

JULY 2016 469

BAY AREA BAY AREA CONTINUED NORTHERN CALIFORNIA CONTINUED CENTRAL VALLEY

Largest AC-335 SAN FRANCISCO: Great Practice! 2100sf, CG-548 ROHNERT PARK: Award winning office by EG-521 FOLSOM Facility: IC-468 SAN JOAQUIN VLY 8ops in desirable location of SF. Call for Details Jim Pride! Collections of $749k in 2015. 1250 sf $50k Price Reduced. All offers considered! $350k $475k w/ 4 ops $545k EG-526 CARMICHAEL IC-572 MODESTO: Broker in AC-566 SAN FRANCISCO: Spectacular views of CN-482 SANTA ROSA: Buy with or without equip- REDUCED: $350k $160k Washington Square. 3ops +2 add’l plumbed in ment. ~ 20 yrs goodwill. $35k ($45k with equip- EG-556 SACRAMENTO: IN-474 STOCKTON: 1400sf office $225k ment) $389k $95k Northern AG-564 SAN FRANCISCO: Over 25 yrs goodwill. DC-476 DUBLIN: Shared Facility. Great for Special- EN-534 ROSEVILLE Facility: IN-506 TURLOCK: Large 5,600+ sf w/ 9 ops near Land’s End ist - Endo, Pedo or Ortho. 1100 sf w/ 2 ops+1 $45k $425k $2.225M add’l $125k EG-560 CARMICHAEL: IN-512 MERCED: California AG-576 SAN FRANCISCO: Part time practice w/ DC-561 SAN JOSE: Eastridge Area, Seasoned $130k Now Only: $110k Amazing Growth Potential. Perfect for 1-3 DDS 4 Staff, Loyal Pts, 6 ops +1 add’l, 2400sf, 10npts/mo EN-558 DAVIS: IN-554 TURLOCK: ops 1.400 sf $550k $300k $650k $795k AN-514 SAN FRANCISCO Facility: Located in the DG-550 SAN JOSE: TWO AMAZING PRACTICES. Call EN-573 SACRAMENTO: JC-541 FRESNO Facility bustling financial district! 1,007 sf w/4 ops. Re- for Details! $1.4M $93.1k Call for Details! Extensive Buyer duced to $125k! DN-497 PLEASANTON Facility: Great Location! FC-334 NORTHERN CA: JG-491 FRESNO: AN-565 SAN FRANCISCO: This remarkable oppor- 870 sf w/ 3 ops + 1 add’l. Owner Financing $480k Real Estate Also Available! REDUCED! $395k Database & tunity could be your “dream come true”! 2,067 sf w/10% Down! Reduced! $95k FC-415 FT. BRAGG: JN-551 COALINGA AREA: w/ 6 ops. $1.05M DG-519 SANTA CLARA Facility: Move In Ready! $425k REDUCED! $395k! Unsurpassed BC-361 OAKLAND: Established for over 23+ years! 2240 sf w 6 fully equipped ops $225k FC-489 CLEARLAKE: 2,200 sf w/ 7 ops. Seller is retiring. Now Only: DG-547 SUNNYVALE: 900sf w 3 ops & Priced to $470k / 50% interest in RE Also Available SPECIALTY PRACTICES $330k sell at only $195k - Call for Details! FN-527 TRINITY COUNTY: “Pride Instute” Exposure allows BC-509 SAN LEANDRO: Facility Only, 800 sf, 3ops DN-542 FREMONT Facility: Spacious & beauful- designed! $250k BC-544 ALAMEDA COUNTY Pedo: w/ xray in each op. Call for Details $60k ly equipped State-of-the-Art! 3,400 sf w/ 5 ops + GC-472 ORLAND: $225k us to offer you BC-520 HAYWARD Facility: Located in Down- 4 add’l. $295k $160k BG-517 NORTH EAST BAY Endo: town, 1500 sf, 4 equipped ops, X-Rays in 3 ops. DN-557 SALINAS: 3,000 sf w/ 7 ops and collecng GG-386 REDDING: $500k Call for Details $65k over $2.225M. Priced at only $1.4M ONLY $260k CC-346 SO MARIN CO Perio: No reasonable

BC-432 PITTSBURG: Own this family-oriented GG-453 CHICO: $325k offer will be refused! Reduced $150k Practice! 1,640 sf w/ 6 ops. Seller is Retiring NORTHERN CALIFORNIA GG-454 PARADISE: CG-424 NAPA Prostho: $350k $525k BC-549 LAMORINDA AREA Facility: Excellent EC-525 SACRAMENTO: Great Location! Excellent GN-244 OROVILLE: $690k Location! Highly Visible, 900sf w/ 3ops +1 Visibility! 1,500 sf w/ 3ops, 10-15 new pts/mo Only $315k DC-459 SF PENINSULA Perio: plumbed add’l. Reduced $75k $220k GN-399 REDDING: $580k BN-504 RICHMOND: Established Practice and EC-531 GREATER SACRAMENTO: Pracce and $150k FN-536 LAKE COUNTY Pedo: Real Estate! 1,450 sf w/ 2 ops + 2 add’l $100k / Real Estate for Sale! 1,750sf w/ 4ops + 1 add’l, GN-507 CHICO: before Now Only: $275k! RE $700k 8npts/mo $800k Practice $535k Real Estate $750k IC-543 CENTRAL VALLEY Ortho: BN-505 CONCORD Facility: The essence of com- EN-464 ROCKLIN Facility: Don’t miss out on this GN-546 CHICO AREA: $180k fort and funconality. 800 sf w/ 3ops. Now remarkable opportunity! 2,150 sf w/ 4 ops. $350K JC-540 FRESNO Sleep Apnea Only: $30k! Now Only: $100k HC-461 SONORA: Call for Details! CC-567 ST. HELENA: Live and Practice in beautiful EN-475 ROSEVILLE Facility: Hesitate and you Practice $700k & RE Also Available! Wine Country, 5ops in 1842sf, single-story bldg. might miss out on this opportunity! 875 sf w/ 2 HN-213 ALTURAS: $890k ops + 2 add’l. Now Only: $25k! $115k CG-537 MARIN COUNTY: Rare Opportunity in EG-479 FOLSOM: History is alive here with trib- HN-280 NO EAST CA: $60k upscale, highly desirable area. State of the art utes to the past! 1,600 sf w/ 3ops.. $225k HN-290 PLACERVILLE: Excellent Merger Op! office. 2400 sf w/ 7 ops $1.1M EN-484 FOLSOM Facility: Fantastic Turn-Key Op- $210k portunity! Come live, practice and grow here! HN-539 Central Sierra/Tuolumne Co: 1,934 sf w/ 4 Ops. Now Only $125k $175k

800.641.4179 [email protected] Timothy Giroux, DDS Jon B. Noble, MBA Mona Chang, DDS John M. Cahill, MBA Edmond P. Cahill, JD

BAY AREA BAY AREA CONTINUED NORTHERN CALIFORNIA CONTINUED CENTRAL VALLEY

Largest AC-335 SAN FRANCISCO: CG-548 ROHNERT PARK: EG-521 FOLSOM Facility: Stands out above the rest! Don’t Miss this IC-468 SAN JOAQUIN VLY: High-End Restore Pracce! 2500+sf, 6 ops, one! 1,200 sf w 3 ops. Well Equipped! $50k Price Reduced. All offers considered! $350k $475k $545k EG-526 CARMICHAEL: Relocating and leaving 30yrs Goodwill behind! IC-572 MODESTO: In desirable Dental/Medical Professional building of Broker in AC-566 SAN FRANCISCO: CN-482 SANTA ROSA: 1350 sf w/ 4ops & opt to grow! REDUCED: $350k town, 3ops in 1300sf office. $160k $35k ($45k with equip- EG-556 SACRAMENTO: Near CSUS Campus. Long-term 2nd generation IN-474 STOCKTON: Too good to be true? Absolutely not! 1,600 sf w/ 3 $225k ment) office. 935 sf w/ 4 ops $389k ops. $95k Northern AG-564 SAN FRANCISCO: DC-476 DUBLIN: EN-534 ROSEVILLE Facility: Locaon, Locaon, Locaon! Turn-key…just IN-506 TURLOCK: Practice in the heart of the Central Valley! 2,000 sf needs you! 2,000 sf w/4 ops. $45k w/ 5ops + 1 add’l. $425k $2.225M $125k EG-560 CARMICHAEL: Focusing on the philosophy of treang paents IN-512 MERCED: This immaculate practice is an absolute jewel! 1,200 California AG-576 SAN FRANCISCO: DC-561 SAN JOSE: as family! 1,200 sf w/ 3 ops + 1 add’l. $130k sf w/ 4ops + 1 add’l. Now Only: $110k EN-558 DAVIS: Designed for maximum office efficiency and paent IN-554 TURLOCK: A small town feel but with “big city” amenies! 1,900 $550k $300k flow! 1,487 sf w/ 4 ops + 1 add’l. $650k sf w/ 5ops. $795k AN-514 SAN FRANCISCO Facility: DG-550 SAN JOSE: EN-573 SACRAMENTO: The goal and focus of this pracce is to provide JC-541 FRESNO Facility: 1,210 square feet and consists of 2 fully Re- $1.4M excellent service! 1,075 sf w/ 2 ops. $93.1k equipped ops and plumbed for add’l op Call for Details! Extensive Buyer duced to $125k! DN-497 PLEASANTON Facility: FC-334 NORTHERN CA: Emphasis on prevention. 1,200 sf w/ 4 ops JG-491 FRESNO: Well-established. 40-50 new Pt/mo. 1,452 sf w/ 4 AN-565 SAN FRANCISCO: Owner Financing $480k / Real Estate Also Available! fully equipped ops. REDUCED! $395k Database & w/10% Down! Reduced! $95k FC-415 FT. BRAGG: Excellent Practice! Dr. avgs 18+ pts/day & 20+ npts/ JN-551 COALINGA AREA: Serving this community of working families! $1.05M DG-519 SANTA CLARA Facility: mo, 1,800 sf w/ 5 ops + 1 hyg. Op $425k Paperless Pracce. 1,200 sf w/ 3 ops. REDUCED! $395k! Unsurpassed BC-361 OAKLAND: $225k FC-489 CLEARLAKE: Located on “4-Corners” of Hwy 53, 4ops in shared Now Only: DG-547 SUNNYVALE: 3600sf facility. $470k / 50% interest in RE Also Available SPECIALTY PRACTICES $330k $195k - Call for Details! FN-527 TRINITY COUNTY: Be the only dentist in town! “Pride Instute” Exposure allows BC-509 SAN LEANDRO: DN-542 FREMONT Facility: designed! 2350sf w/ 5 ops +1 add’l. $250k BC-544 ALAMEDA COUNTY Pedo: 1,056sf w/ 4 chairs in growing, revi- $60k GC-472 ORLAND: Live & Practice in charming small town community. talized community, Seller Retiring $225k us to offer you BC-520 HAYWARD Facility: $295k 1,000 sf w/ 2ops. Seller Retiring. $160k BG-517 NORTH EAST BAY Endo: 2,750 sf w/ 8 ops! Strong Practice! DN-557 SALINAS: GG-386 REDDING: Amazing Practice. Lease or Buy Real Estate! 2,860 sf $500k $65k Priced at only $1.4M w/ 4 ops. Plumbed for 2 add’l! ONLY $260k CC-346 SO MARIN CO Perio: Beautiful 1,142 sf w/ 3 ops. No reasonable

BC-432 PITTSBURG: GG-453 CHICO: 5,000 sf w/ 7 ops Perfect for 1 or more dentists! $325k offer will be refused! Reduced $150k NORTHERN CALIFORNIA GG-454 PARADISE: ~2,550 sf w/ 9 ops. 40 yrs goodwill! Amazing Oppor- CG-424 NAPA Prostho: Office has Digital X-ray & NEW 3D Imaging $350k tunity! $525k Unit! Ready for Experienced, high-end Prosthodontist! On track to col- BC-549 LAMORINDA AREA Facility: EC-525 SACRAMENTO GN-244 OROVILLE: Must See! Gorgeous, Spacious. 2,500 sf w/5 ops! lect just under $1m $690k Collections over $450k in 2013. Only $315k DC-459 SF PENINSULA Perio: 50% Partnership Buy In! Call for Details! Reduced $75k $220k GN-399 REDDING: Loyal patient base and relaxed workweek schedule. $580k BN-504 RICHMOND: EC-531 GREATER SACRAMENTO: Pracce and 1,440 sf w/3 ops. $150k FN-536 LAKE COUNTY Pedo: Focusing on Prevent dental problems $100k / Real Estate for Sale! GN-507 CHICO: It just doesn’t get any better than this! 3,000 sf w/ before they begin! 1,750 sf w/3 ops. Now Only: $275k! RE $700k $800k 7ops. Practice $535k Real Estate $750k IC-543 CENTRAL VALLEY Ortho: 1,650 sf w/ 5 chair bays & plumbed for BN-505 CONCORD Facility: EN-464 ROCKLIN Facility: GN-546 CHICO AREA: Well-known for offering quality denstry with 2 add’l, Strong Refs & Satisfied Pts Base $180k Now sedaon. 2,600sf w/ 4 ops. $350K JC-540 FRESNO Sleep Apnea: Movated Seller rering! Step right in Only: $30k! Now Only: $100k HC-461 SONORA: In the beautiful Sierra Foothills, 4ops, 1350sf, free- and make yours! Call for Details! CC-567 ST. HELENA: EN-475 ROSEVILLE Facility: standing bldg.. Practice $700k & RE Also Available! HN-213 ALTURAS: This well managed pracce connues to have con- $890k Now Only: $25k! sistent revenues! 2,200 sf w/ 3 ops + 1 addil $115k CG-537 MARIN COUNTY: EG-479 FOLSOM HN-280 NO EAST CA: Only Practice in Town 900 sf w/ 2 ops $60k . $225k HN-290 PLACERVILLE: Excellent Merger Op! FFS. 1,400 sf w/ 4 ops $1.1M EN-484 FOLSOM Facility: Fantastic Turn-Key Op- $210k portunity! HN-539 Central Sierra/Tuolumne Co: The perfect Merger Op in a rural Now Only $125k Sierra Community! 2,000 sf w/ 5 ops. $175k

“ASK THE BROKER” CAN NOW BE FOUND AT WWW.WESTERNPRACTICESALES.COM Dr. Bob CDA JOURNAL, VOL 44, Nº7

A Dentist’s Guide to Fitness

You didn’t think for a minute, The following Dr. Bob column was Fountain of Youth, fi nally fi nds it just moron-like, that you were going originally printed in the February 1996 off I-95 and approaches the attendant issue of the Journal. for permission to bathe. “No problem,” to sit in a high-back leather says the Seminole-in-charge (sic), chair issuing orders, giving The human body. This God-given handing him a towel. “Fifty pesos and temple of the soul. Treasure it from birth, don’t forget to take off your …” dictation and doing three- nourish it with oat bran and wheat germ, But Ponce, eager to enjoy the martini lunches, did you? stoke it with vitamins and minerals, fruits of eternal youth, jumps in still baste it with Oil of Olay, this marvelous clad in his armor and plummets machine. Give it a massage and aerobic to the bottom like a safe. exercises, treat it to Nautilus and every Well, what did you expect? When you Robert E. other body-enhancing gadget and potion signed up for dental school, they didn’t from the fertile minds of man and mention the nature of the work? C’mon, Horseman, Elizabeth Arden, then protect it from everybody knows about the postural and DDS every kind of stress you can and you know visual defects that set in about the second what? — that sucker will die anyway. semester and go downhill from there. You ILLUSTRATION It’s ironic, learning that Ponce de didn’t think for a minute, moron-like, BY VAL B. MINA Leon, after years of searching for the that you were going to sit in a high-back

472 JULY 2016 CDA JOURNAL, VOL 44, Nº7

leather chair issuing orders, giving dictation It became apparent just after the thing to do. That’s why the scoring was and doing three-martini lunches, did you? game was invented that running altered to jump from 15 to 40 just to get the No, it’s our lot in life to give new game over with as fast as possible. Dentists meaning to the biblical phrase “laying around in the hot sun chasing a little are advised to take up ping pong, which is on of hands.” To do this requires ball was a dumb thing to do. That’s the adult version of tennis without the sun. that we get fi t and remain so even if Scuba diving. Younger dentists who it means eventually questioning the why the scoring was altered to miss the excitement of doing wheelies validity of the whole concept. jump from 15 to 40 just to get the on a motorcycle with no hands, no Initiated by dentists still young and helmet and no brains or racing around naïve enough to think they could reverse game over with as fast as possible. in their fathers’ Oldsmobiles without this deterioration, the ongoing fi tness craze the benefi t of functioning thought that has gripped this country for the past processes, frequently take up scuba couple of decades shows no sign of abating. diving as an antidote to the deadly Orthopedic offi ces throughout the nation Golf does not contribute to your fi tness confi nement of the operatory. are littered with shin splints, torn tendons but forces you to recite long, boring Unless you live in Tahiti, Hawaii, the and sorely abused bodies. What we’ve got anecdotes whose tediousness is exceeded Bahamas or the Grand Caymans, you will to do is sort out the things that will cripple only by the tales of your fellow golfers. experience only grinding regret every time or maim us, thus making us ex-dentists, Bowling. Forget bowling, too. Using three you look into your closet at the jillions of and seek those things that will give us a of your vital fi ngers to propel a large leaden dollars’ worth of wetsuits, regulators, masks, better chance of fulfi lling our destinies. sphere in an effort to fl atten 10 objects fi ns, booties, hoods, gloves, depth gauges, The following information just so you can do it again and again to the tanks and special wrist watches the size of a will not help a bit: accompaniment of gawdawful noise makes manhole cover with 89 functions, none of Right off the bat, so to speak, that no sense at all. The requisite costume is an which reveal the correct time, getting dusty eliminates baseball, as it is one of the embarrassment from the garish shoes to the from disuse. I realize that to a dedicated sports invoking the use of hands, our most sports shirts with embroidered advertising for scuba diver this is all offset by the sight of productive appendages, or at least the ones brake relinings or fast food joints. Bowling a fi sh going by in its element deep in the we’re most interested in here. Lose the use would be bad enough if you did it alone, but murky depths of water two degrees above of your hands and what’s left for you? — tradition requires that you do it in concert freezing, but to a rational person, 30 minutes nothing but the lecture circuit, a scam that’s with a bunch of bozos whose motivation of a Jacques Cousteau rerun should suffi ce. already been thought of by hundreds of your is based entirely on getting a night out. Skiing. You would think that a colleagues to the point where in a few years Tennis. Tennis used to be a gentleman’s dentist, even a young one without there will be nobody left to do the actual game. From the crisp, white, impeccable the wisdom that comes from years of work; they’re all out lecturing to each other. outfi ts to the strict adherence to manners listening to patients lie about their So let’s see what might contribute and tradition, it seemed to fulfi ll all the fl ossing habits, would see the idiocy of to your fi tness program without the requirements. Even though the dominant falling totally out of control down the danger of forcing you out to tell other forearm risked resembling Popeye’s while face of a mountain while wearing 7-foot dentists how to bleach teeth. the other one remained like Olive Oyl’s, boards strapped to boots the size of Golf. Golf gets you into the open air, it was still a fairly civilized sport. This has microwave ovens. Boards that he waxes and it’s where patients think you are on changed. If you’ve watched tennis lately, you to make him fall even faster, for God’s Wednesdays, anyway, but that’s about know that Andre Agassi, with his sartorial sake. Well, lots of dentists do this. You all you can say for golf. To participate, statement is now a role model; a tennis ball can observe these dentists at the resort it is necessary to humiliate yourself by traveling at speeds in excess of 120 mph can bars bemoaning their rotator cuffs and wearing ridiculous pants and impractical alter your physical well-being forever; and spiral fractures. It’s the only sport open shoes whose only other use is tenderizing there is way too much sweating and grunting. to dentists that requires more expensive cheaper cuts of meat. You sit on your Umpires risk getting a biff in the snoot for gear than scuba diving. The cost of dental stool, you sit in your golf cart. You a bad call, and people have been known to stuff needed for a family of four just to try to get a small object into a hole, and get stabbed. It became apparent just after the get to the falling-down stage of skiing you have a level of frustration as high or game was invented that running around in exceeds the S&L bailouts. Skiing does higher than that in your dental practice. the hot sun chasing a little ball was a dumb not contribute to fi tness, it cancels it.

JULY 2016 473 JULY 2016 DR. BOB

CDA JOURNAL, VOL 44, Nº7

Jogging. Nobody jogs anymore. I just That doesn’t leave us with much. chuckling dealer who sold them to you. put that in there so that if you are still Certainly dentists with IQs superior to Leave them in the closet, under the bed jogging based on the misguided idea that paramecia should eschew pumping iron. with the dust bunnies or in the garage it’s doing you some good, you’ll know it’s That branch of insanity will instantly where they have resided peacefully since time to quit. Power walking is where it’s at identify any aneurysm you may have in your the second week they were brought home now. With your $125 special cross-training body, besides running the risk of making with such high hopes and fi rm resolutions. walking shoes that has Korea giggling you appear to have had badly managed Above all, don’t be too hard on all the way to the bank, you’ll discover silicone injections infl icted on areas where yourself for being such an incredible jerk power walking is to plain strolling what you don’t even have muscles and never did. for getting in such bad shape in the fi rst St. Vitus’ Dance is to Barcalounging. I know it’s too late to save you from place. And if you decide to take up one If you insist on power walking, do it the mistake of buying the stationary of these “fi tness” sports in spite of all the in the early hours or after dark so that bicycle, rowing machine, treadmill and intelligence to the contrary, be sure to check passing motorists won’t lose control with the like. The only benefi t these machines with your physician before you start. You laughter and crash into light poles. have ever produced accrued to the know what kind of great shape he’s in. ■

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