Expected Double Digit Growth for Invisible Braces Through 2021 

Invisible braces have made orthodontic treatment an attractive option for many consumers. Improvements in their technology and growing awareness of their aesthetic applications have led to their success in the global market, which will continue to rise at a 12.16% compound annual growth rate (CAGR) through 2021, reports Azoth Analytics. The market already has a strong foothold in North America and Europe. Growing dental tourism in Mexico and Thailand, though, will continue to contribute to its success. India and Brazil along with emerging nations in Latin America and the Asia-Pacific region also will fuel this new growth. Specifically, in China, Invisalign holds 35% of the market, while indigenous brand Angel Align leads with 38%, according to Research and Markets. Medical reforms, growing per capita disposable income, and greater public health awareness are driving dentistry’s growth in the nation. Overall, China’s dental apparatus and services market will grow from about $6.1 billion in 2015 to an expected $22.8 billion in 2020. The global dental market— which includes preventive, restorative, implants, prosthetics, orthodontics, endodontics, and dental equipment—will surpass $50 billion by 2020, Research and Markets reports. - See more at:  http://www.dentistrytoday.com/news/industrynews/item/991-invisible-braces-to-see-12-16-growth-through-2021?highlight=WyJicmFjZXMiLCJicmFjZXMnIl0=

Sourch: Dentistry.com News

APRIL IS NATIONAL FACIAL PROTECTION MONTH

Many sports injuries can be prevented by wearing appropriate protective gear.  April is National Facial Protection Month.  Dr. Kaprelian wants to remind athletes, their parents and coaches to play it safe by wearing mouth guards and appropriate protective gear when participating in activities that could cause injuries.

Mouth guards are one of the least expensive pieces of protective equipment available.  Over-the-counter versions may cost as little as $5.  Mouth guards can protect teeth and jaws, but they only provide protection when they are worn, so Dr. Kaprelian advises parents and coaches to remind youngsters to always use a mouth guard when participating in any activity during which the mouth could come into contact with a hard object or the pavement. 

Athletes who wear braces should consult their orthodontist for a recommendation of the best mouth guard to wear during orthodontic treatment,

Consistent use of other protective equipment is important, too.  Helmets save lives and prevent head injuries.  They should be worn for activities such as bicycling, skateboarding or skating on in-line skates.  Helmet wear is mandated for many organized sports.  Helmets should be worn for any activity that puts the head at risk. 

Face guards, devices made of plastic or metal that attach to baseball helmets, help to prevent facial injuries as well.

National Facial Protection Month is co-sponsored annually by the American Association of Orthodontists (AAO), the oldest and largest dental specialty organization in the world, and by the American Association of Oral and Maxillofacial Surgeons (AAOMS), the American Academy of Pediatric Dentistry (AAPD), Academy for Sports Dentistry (ASD)and the American Dental Association (ADA).

Orthodontists receive an additional two to three years of specialized education beyond dental school to learn the proper way to align and straighten teeth.  Only those with this formal education may call themselves “orthodontists,” and only orthodontists are eligible for membership in the AAO.  The AAO’s website is mylifemysmile.org.

Mouth Piercings Increase the Risk of Tooth Fractures and Gum Disease

7/07/08

According to a study conducted by Liran Levin, DDS, a dentist from the Department of Oral Rehabilitation in the School of Dental Medicine at Tel Aviv University, teens with oral piercings are at high risk for tooth fractures and gum disease.

“There are short-term complications to piercings in low percentages of teens, and in rare cases a piercing to the oral cavity can cause death,” said Levin. “Swelling and inflammation of the area can cause edema, which disturbs the respiratory tract.”

Levin warns that the most common concerns—tooth fracture and periodontal complications—are long-term.

“There is a repeated trauma to the area of the gum,” said Levin. “You can see these young men and women playing with the piercing on their tongue or lip. This act prolongs the trauma to the mouth and in many cases is a precursor to anterior tooth loss.”

In the study, the researchers surveyed teens with and without piercings and asked them a number of questions about their oral health, their knowledge of the risk factors associated with piercings, and about their piercing history, before conducting the clinical oral exams.

Levin noted that the youngsters who opted for oral piercings were very concerned about body image, but seemed to be unaware of the future risks that they can cause.

According to Levin and his colleagues, teens should avoid getting their mouths pierced. If the teen insists, then it’s essential that piercing tools are disposable, and that all other equipment is cleaned in an onsite autoclave to help reduce infection. After the procedure, the area should be rinsed regularly with a chloroxidine-based mouthwash for 2 weeks. The teen should also avoid playing with the piercing and clean it on a regular basis. Calculus deposits on the piercing may form over time and should be removed by a dentist. Checkups should be made regularly.

[Science Daily, June 24, 2008]

Quantification and Qualification of Bacteria

Trapped in Chewed Gum

Stefan W. Wessel1, Henny C. van der Mei1*, David Morando2, Anje M. Slomp1, Betsy van

de Belt-Gritter1, Amarnath Maitra2, Henk J. Busscher1

1 University of Groningen and University Medical Center Groningen, Department of Biomedical Engineering,

Groningen, The Netherlands, 2 William Wrigley, Jr. Company, Chicago, Illinois, United States of America

* h.c.van.der.mei@umcg.nl

Abstract

Chewing of gum contributes to the maintenance of oral health. Many oral diseases, including

caries and periodontal disease, are caused by bacteria. However, it is unknown whether

chewing of gum can remove bacteria from the oral cavity. Here, we hypothesize that chewing

of gum can trap bacteria and remove them from the oral cavity. To test this hypothesis,

we developed two methods to quantify numbers of bacteria trapped in chewed gum. In the

first method, known numbers of bacteria were finger-chewed into gum and chewed gums

were molded to standard dimensions, sonicated and plated to determine numbers of colony-

forming-units incorporated, yielding calibration curves of colony-forming-units retrieved

versus finger-chewed in. In a second method, calibration curves were created by fingerchewing

known numbers of bacteria into gum and subsequently dissolving the gum in a

mixture of chloroform and tris-ethylenediaminetetraacetic-acid (TE)-buffer. The TE-buffer

was analyzed using quantitative Polymerase-Chain-Reaction (qPCR), yielding calibration

curves of total numbers of bacteria versus finger-chewed in. Next, five volunteers were requested

to chew gum up to 10 min after which numbers of colony-forming-units and total

numbers of bacteria trapped in chewed gum were determined using the above methods.

The qPCR method, involving both dead and live bacteria yielded higher numbers of retrieved

bacteria than plating, involving only viable bacteria. Numbers of trapped bacteria

were maximal during initial chewing after which a slow decrease over time up to 10 min was

observed. Around 108 bacteria were detected per gum piece depending on the method and

gum considered. The number of species trapped in chewed gum increased with chewing

time. Trapped bacteria were clearly visualized in chewed gum using scanning-electron-microscopy.

Summarizing, using novel methods to quantify and qualify oral bacteria trapped

in chewed gum, the hypothesis is confirmed that chewing of gum can trap and remove bacteria

from the oral cavity.

Introduction

Descriptions of the first use of chewing gum date back to the ancient Greek, who used tree resins

from the mastic tree to quench thirst and refresh their breath. The first commercial chewing

gum was not successfully marketed until the late 19th century, when the rubbery tree sap of the

Sapodilla tree formed the basis for gum manufacturing [1]. In the late 20th century, chewing

gum is not only regarded as a symbol of lifestyle, but also effects on cognitive performance,

mood, alertness and appetite control have been reported [2–5]. Moreover, chewing gum has

developed more and more towards an oral care and functional food product (“nutraceutical”),

as it provides an easily applicable drug delivery vehicle with potential benefits for oral health

[1]. High consumption rates, up to 2.5 kg per person per year, have made it into a billion dollar

industry [6,7]. Most chewing gums consist of a mixture of food grade synthetic elastomers, like polyvinyl

acetate or polyisobutylene, generally referred to as the gum-base [1]. Important requirements

to gum-base materials are that they do not dissolve in the oral cavity and can be chewed for

long periods of time without undergoing compositional and structural changes. In most commercially

available chewing gums, the gum-base is supplemented with sweeteners, flavors and

other bulking agents, while nowadays sugar is frequently replaced by artificial sweeteners such

as sorbitol, xylitol or mannitol [6,7].

The inclusion of xylitol and other artificial sweeteners has been described to reduce the formation

of oral biofilms on teeth [8,9]. Oral biofilms are causative to the world’s most widespread

infectious diseases, namely dental caries and periodontal disease [10]. Caries arises

from an unbalance between naturally occurring de- and remineralization of dental enamel.

Demineralization occurs when the pH of oral biofilm drops below 5.5 [11] due to the fermentation

of carbohydrates by specific bacterial strains in oral biofilms on teeth. Most artificial sugars

are not or barely fermented by oral bacteria and therewith do not lower the pH [12]. Moreover,

chewing gum yields enhanced mastication that stimulates salivation, which clears fermentable

carbohydrates, dislodges loosely bound oral bacteria from oral surfaces [13] and increases the

concentrations of calcium and phosphates in the oral cavity required for remineralization [14].

Fluorides have been added to commercial gums to prevent enamel demineralization and stimulate

remineralization [15]. It is tempting to regard the chewing of gum as an addendum to

daily oral hygiene procedures, especially since most people are unable to maintain a level of

oral biofilm control required to prevent disease through daily toothbrushing and other conventional

oral hygiene measures. This has led to the incorporation of antimicrobials like chlorhexidine

[16] and herbal extracts [17] to chewing gums and gums have indeed been demonstrated

successful in preventing re-growth of oral biofilm [18]. It is also known that chewing of gum

aids in the removal of interdental debris [19]. To increase the cleaning power of chewing gum,

detergents like polyphosphates [20] have been added to gums. However, it is unclear whether

chewing of gum itself will actually remove bacteria from the oral cavity. Especially the preferential

removal in sizeable numbers of disease-causing microorganisms like acid-producing Streptococcus

mutans or species that are regarded as initial colonizers of tooth surfaces by chewing

gum would turn chewing gum into a valuable addendum to daily oral hygiene.

Therefore, the aim of this study is firstly to develop methods to quantify the number of bacteria

that are trapped into a gum after chewing, and secondly to qualitatively determine the

bacterial composition of bacteria trapped in chewed gums. The first method is based on measuring

the number of colony-forming units (CFUs) that can be retrieved from pieces of gum,

chewed by different volunteers. The method relies on finger-chewing known numbers of different

oral bacterial strains into commercially available spearmint gums and retrieving bacteria

from the gums by sonication followed by agar-plating of the bacterial suspension to yield a

Bacterial Trapping in Chewed Gum calibration curve. By comparing it to the number of bacteria retrieved from pieces of

gum chewed by volunteers, the number of CFUs trapped in pieces of chewed gum can be calculated.

In the second method, pieces of chewed gum are dissolved and the amount of bacterial genomic

DNA is quantitated using quantitative Polymerase-Chain-Reaction (qPCR) and converted

to numbers of bacteria trapped in the chewed gums using a calibration curve, also obtained by

finger-chewing. The composition of the different bacterial species trapped in chewed gum was

compared with the composition of the salivary microbiome and the microbiome adhering to

teeth using Denaturing Gradient Gel Electrophoresis (DGGE). Finally, we demonstrate bacterial

presence in chewed gum using Scanning Electron Microscopy (SEM).

Materials and Methods

Chewing Gum

Two commercially available spearmint chewing gums were used in this study: Gum A – (commercially

available spearmint gum, 1.5 g tabs). Composition in descending order of predominance

by weight: Sorbitol, gum base, glycerol. Natural and artificial flavors; less than 2% of:

Hydrogenated starch hydrolysate, aspartame, mannitol, acesulfame K, soy lecithin, xylitol,

beta-carotene, blue 1 lake and butylated hydroxytoluene.

Gum B – (commercially available spearmint gum, 1.5 g tabs.). Composition in descending

order of predominance by weight: Sorbitol, gum base, glycerin, mannitol, xylitol. Natural and

artificial flavors; less than 2% of: Acesulfame K, aspartame, butylated hydroxytoluene, blue 1

lake, soy lecithin and yellow 5 lake. Both gums were similarly hydrophobic with water contact

angles on sectioned pieces of gum of 69 and 74 degrees for gum A and B, respectively.

Method 1: Enumeration of Bacteria Trapped in Chewed Gums using

Sonication of Gum Molded to Standard Dimensions

Basics of the Method and Preparation of a Calibration Curve

In this method, four different bacterial strains were used for the preparation of a calibration

curve that relates the numbers of CFUs retrieved from a piece of gum to the numbers of CFUs

incorporated in the gum for coccus-shaped Streptococcus oralis J22, Streptococcus mutans

ATCC 25175, Streptococcus mitis ATCC 9811 and rod-shaped Actinomyces naeslundii T14VJ1.

S. oralis and A. naeslundii are considered initial colonizers of tooth surfaces in vivo [21,22],

while S. mutans is causative to dental caries [23] and S. mitis is an abundantly present species

in the oral cavity [24]. Streptococci were grown aerobically in Todd Hewitt Broth (THB) at

37°C and actinomyces anaerobically in Schaedler broth. Bacteria were first grown on THB agar

or blood agar plates from a frozen stock in dimethylsulfoxide for 24 h after which one colony

was inoculated in 10 ml of the appropriate culture medium and incubated for 24 h. A main culture

was prepared with a 1:10 dilution in fresh medium for 16 h. Main cultures were sonicated

for 1 × 10 s at 30W(Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT, USA) to

suspend bacterial aggregates. The bacterial concentration was determined using the Bürker

Türk counting chamber, while percentage viability of the suspended bacteria was determined

after serial dilution and agar-plating. Next, concentrations were adjusted to 104, 105, 107 and

109 bacteria per ml. Since viability of the cultures was near 100%, these numbers are equivalent

to 4, 5, 7 and 9 log-units of CFUs per ml.

For each strain, known numbers of CFUs were finger-chewed into gum pieces by adding

1.5 g chewing gum together with 200 μl of a bacterial suspension into the finger of a sterile

latex glove (Powder-Free Latex Examination Gloves, VWR international, Radnor, USA). Next,

bacteria were finger-chewed into the gumin a water bath at 37°C for 5min. After finger-chewing,

Bacterial Trapping in Chewed Gum the gum was removed from the glove, dipped once in 10 ml sterile water and put

into a Teflon mold (15 × 15×1mm) with a sterile pair of tweezers to create reproducible gum dimensions (15 ×

15 × 4 mm) and surface area (690 mm2). Subsequently, the gum was inserted in sterile polystyrene

cups with 5 ml filter sterile Reduced Transport Fluid (RTF) [25]. Bacteria were removed from the

gum surface layer by sonication for 60 s in a water bath sonicator (ELMA Transsonic TP690, Elma

GmbH & Co, Germany). Sonication times up to 60 s did not affect bacterial viability [26,27].

Finally, the resulting suspension was serially diluted, plated on THB agar or blood agar plates

(Blood agar base no. 2, 40 g/l, hemin5mg/l,menadion 1 mg/l, sheep blood 50 ml/l) and incubated

at 37°Cfor 48 h after which the number of CFUs retrievedwere counted.Accordingly, since different

numbers of bacteria were finger-chewed into the gums, a calibration curve was made of the

numbers of CFUs retrieved from each gum for the different bacterial strains versus the numbers of

CFUs finger-chewed into the gum. To account for possible loss of bacteria due to adhesion to the

inner surface of the glove, the glove finger was turned inside out after removal of the gum and

sonicated in 10ml filter sterileRTF for 60 s and serial dilutions plated on agar plates as described

above after which the number of CFUs lost were determined. Similarly, the water in which the

finger-chewed gums were dipped (see above) was analyzed for bacterial losses. Calibration curves

were made in triplicate for each chewing gum and bacterial strain.

Application of the Method in Human Volunteers

Volunteers included in this study were five healthy members of the department of Biomedical

Engineering (1 male, 4 females, aged 27 to 56 years). All experiments were performed according

to the rules as set out by the Medical Ethics Committee of the University Medical Center

Groningen, and they approved this study (approval METc 2011/330). Volunteers gave their

written informed consent. Inclusion criteria described that all volunteers should be in good

health and have at least 16 natural elements. Exclusion criteria were the use of antibiotics or

mouth rinses in the month prior to the study or the use of antibiotics, mouth rinses and additional

chewing gum during the study. Furthermore, volunteers were requested to brush their

teeth twice a day, according to their habitual routines.

On separate days, volunteers were asked to chew 1.5 g (one serving size) of each chewing

gum once a day at the same time for 0.5, 1, 3, 5 or 10 min according to their own personal routine

without specific instructions for chewing. Chewing time and gum types (A or B) were randomly

assigned to the volunteers over the experimental period. After chewing, the gum was

spit in a polystyrene cup with 10 ml sterile water, after which the chewed gum was put into the

Teflon mold and sonicated, as described above. Resulting suspensions were serial diluted, agarplated

and the numbers of CFUs were determined after incubation for 7 days at 37°C under anaerobic

conditions (5% H2, 10% CO2, 85% N2) (Concept 400 anaerobic workstation, Ruskinn

Technology Ltd., Pencoed, UK). Finally, the numbers of CFUs retrieved from the gums after

different chewing times and for both types of gum were converted to the total number of CFUs

trapped in chewed gums using the calibration curve obtained from finger-chewing known

numbers of bacteria into the gums. Note that this requires the assumption that bacterial viablility

is equally maintained in finger-chewed gum as in gum chewed by volunteers. All experiments

were carried out in duplicate for each volunteer, gum type and time point.

Method 2: Enumeration of Bacteria Trapped in Chewed Gums using

qPCR and Microbial Composition

Basics of the Method and Preparation of a Calibration Curve

Similar to method 1, a calibration curve was made by finger-chewing known numbers of S. oralis

J22, S. mutans ATCC 25175, S. mitis ATCC 9811 or A. naeslundii T14V-J1 in the different

Bacterial Trapping in Chewed Gum

PLOS ONE | DOI:10.1371/journal.pone.0117191 January 20, 2015 4 / 12

spearmint gums. Bacterial concentrations were adjusted using the Bürker Türk counting chamber

to 107, 109 and 1010 bacteria per ml, in which the latter concentration was achieved by centrifugation

(5 min, 5000 g at 10°C). After finger-chewing as described above, the gum was

removed from the glove, dipped once in 10 ml sterile water and subsequently dissolved in a

mixture of 5 ml chloroform (67-66-3, Fisher Scientific, Waltham, USA) and 3 ml tris-ethylenediaminetetraacetic-

acid (TE) buffer (AM9849, Ambion—LifeTechnologies, Carlsbad, USA) in

a sterile centrifuge tube. The gum was dissolved in 45 min by shaking horizontally. The resulting

suspension was centrifuged for 10 min at 1500 g to remove large particles and gum base

from the aqueous TE buffer top layer.

For qPCR, 17.5 μl master mix was used for every sample consisting of 10 μl PCR—mix (iQ5

SYBR Green Supermix, Bio-rad, Hercules, USA), 5 μl DNA free water (95284, Sigma, St. Louis

MO, USA) and 2.5 μl primer mix (300 nM). To amplify the universal V3 region of the 16S

rRNA gene in all samples F357-GC was used as the forward primer and R-518 [28] as the reverse

primer. In a 384-well PCR plate (HSP-3805, Bio-rad, Hercules, USA ), 2.5 μl of sample dilutions

(1×, 10×, 100×), taken from the centrifuged aqueous TE buffer top layer, was mixed

with 17.5 μl of master mix. Subsequently, a qPCR was performed on a thermocycler (CFX384,

Bio-rad, Hercules, USA), according to a 3 step amplification (95.0°C for 45 s, 58.0°C for 45 s,

72°C for 60 s) of 39 cycles. A calibration curve was obtained by relating threshold cycle (Ct) at

fixed relative fluorescence units to the number of bacteria chewed-in the gum [29,30]. Calibration

curves were obtained for both gums in triplicate for all four bacterial strains. DNA free

water and a piece of unchewed gum, dissolved as described above, were used as

negative controls.

Application of the Method in Human Volunteers

Five healthy members of the department of Biomedical Engineering chewed each type of chewing

gum, as described above. Chewed gum was spit in a polystyrene cup with 10 ml sterile

water after which the gum was dissolved in a sterile centrifuge tube with the mixture of chloroform

and TE buffer. After centrifugation, qPCR was performed using the aqueous TE buffer

top layer (see above). The total number of bacteria trapped in the gum was determined using

the calibration curve. Part of each dissolved gum TE-buffer sample was stored in −80°C for

later DGGE analysis.

Determination of the Bacterial Composition using DGGE

The composition of the different species trapped in pieces of chewed gum was determined

using DGGE and compared to the bacterial compositions of the planktonic, salivary microbiome

and the microbiome adhering to tooth surfaces. After 10 min of chewing, volunteers

were asked to donate 1 ml of unstimulated saliva and collect oral biofilm from their entire dentition

using a cotton swab and a sterile hook in 1 ml RTF. Both saliva and biofilm samples were

centrifuged at 18000 g for 5 min (Eppendorf Centrifuge 5417R, Hamburg, Germany), DNA

was isolated [31], after which the samples were resuspended in 50 μl TE buffer.

The DNA concentration of saliva, biofilm and dissolved gum samples were measured with

the Nanodrop Spectrophotometer (ND-110, NanoDrop Technologies Inc., Wilmington, DE,

USA). A PCR was performed with 100 ng DNA using the primers and amplification program

as described above. The products of the PCR were applied on a polyacrylamide gel (8% w/v) in

0.5 TAE buffer (20 mM Tris acetate, 10 mM sodium acetate, 0.5 mM EDTA, pH 8.3). Using a

100% stock solution (7M urea, 37% formamide) a denaturing gradient was made with the

range of 30–80%. A stacking gel without denaturant was added on top and equal amounts of

sample were applied to the gel. Electrophoresis was performed overnight at 60°C and 120

V. Silver nitrate solution (0.2% AgNO3) was used until maximal staining intensity was reached.

Gels were scanned and transferred to analysis software BioNumerics (v7.1 Applied Maths,

Sint-Martens-Latem, Belgium). Gels were normalized to reference markers that were added to

every gel. Presence of a band on the gel was taken as the presence of a bacterial species or strain

in the sample. The similarity of bands was determined according to the band-based matching

module in the software (0.5% optimization, 1% band tolerance).

Scanning Electron Microscopy

In order to visualize bacteria trapped in chewed gum, a 5 min chewed gum piece was spit into

liquid nitrogen, kept immersed for 2 min and broken into multiple pieces, which were subsequently

examined in a SEM (JEOL JSM-6301F, Akishima, Japan). Gum pieces were fixed directly

for 24 h in 2.0% glutaraldehyde at 4.0°C, washed with 0.1 Mcacodylate buffer and

incubated for 1 h in 1.0% OsO4 in 0.1Mcacodylate buffer at room temperature. After washing

with water, samples were dehydrated with an ethanol series (30, 50 and 70%) each for 15 min

and 3 times 30 min with 100% ethanol. Fracture surfaces of the chewed gum were examined

for the presence of bacteria at a magnification of 7.500× with an acceleration voltage of 2.0 kV

and 39.0 mm working distance.

Statistics

Data was evaluated for normality using Shapiro-Wilk and Kolmogorov-Smirnov test (p < 0.05)

and in case of a normal distribution equality of means was tested using an ANOVA followed by

Tukey-HSD post hoc test (p < 0.05). In case no normal distribution of data was observed, a nonparametric

Kruskal-Wallis test was used (p < 0.05). SPSS v20.0 (IBMCorp., Armonk, USA) to

conduct all statistical analysis.

Results

Bacteria of the four different strains were finger-chewed into the two different types of chewing

gums in order to obtain a relation between the number of bacteria trapped in a gum piece and

the number of CFUs or total bacteria that can be retrieved from a gum by agar-plating or

qPCR, respectively. On average, 0.05 log-units of CFUs were lost due to adhesion to the surface

of the glove in which gums were finger-chewed, while A. naeslundii adhered in slightly higher

numbers to the glove surface than streptococcal strains. Bacterial losses due to dipping the finger-

chewed gum pieces in water were much smaller and amounted on average 0.004 log-units

of CFUs.

Accounting for these losses, linear relations were obtained for both methods (Fig. 1). For

CFUs, the calibration lines were independent of the gum type involved. Lines were generally independent

of the bacterial strains involved, apart from a small but statistically significant difference

(p < 0.05) between A. naeslundii and S. mitis at the highest bacterial concentration

(Fig. 1A). As sonication can only release bacteria trapped in a gum from the outer surface, the

number of bacteria retrieved was roughly 1.5 log-units less than chewed-in. The qPCR method

yielded small but statistically significant differences (p < 0.05) in Ct values for the different

bacterial strains (Fig. 1B). However, neglecting these strain-related differences, average linear

calibration lines could be obtained that were independent of the gum type involved.

Next, volunteers were asked to chew the two types of chewing gums for varying amounts of

time up to 10 min and the number of bacteria chewed-in was determined in terms of CFUs

after sonication and agar-plating or in terms the total number of bacteria, as obtained after dissolving

the gum and performing qPCR on bacterial DNA. Agar plating indicates that most

CFUs are trapped (approximately 7.8 log-units) within the first minute, regardless of the gum

involved, while approximately 1 log-unit less CFUs remained trapped in a gum piece after

Bacterial Trapping in Chewed Gum prolonged chewing (Fig. 2A). qPCR yields higher numbers of bacteria retrieved than

agar-plating (Fig. 2B), but displays only a minor decrease in total number of bacteria trapped in time for

both types of chewing gums.

The number of species detected in chewed gum increases with increasing chewing time for

both types of chewing gums (Fig. 3A), while after 10 min of chewing 50–70% of the detected

species in the salivary and adhering microbiome are ultimately detected in the chewed gum

piece (Fig. 3B). A more elaborate analysis of the origin of bacterial species found in chewed

gum indicated that 9% and 16% of the species found in chewed gum were solely detected in the

adhering oral microbiome for gum A and B, respectively, while a relatively similar percentage

of approximately 15% of the detected species chewed-in were solely found in the salivary

microbiome (Fig. 3C). Remaining percentages of species found in chewed gum could either be

attributed to the salivary or the adhering microbiome or their origin could not be detected, suggesting

the tongue, gums or oral mucosal surfaces as an origin.

Considering the numbers of bacteria found in chewed gum and the field of view and depth

of focus of SEM, it can be appreciated that microscopic imaging of trapped bacteria in chewed

gum is like looking for a needle in a haystack. Yet after extensive searching, a scanning electron

micrograph could be taken of a chewed gum piece showing an open and porous structure

(Fig. 4) in which trapped bacteria can be observed as direct evidence of the ability of chewing

gum to trap bacteria during chewing.

Figure 2. Bacteria trapped in two different types of spearmint gums chewed by human volunteers as

function of time. The number of bacteria trapped in chewed gums for two types of spearmint gums as a

function of the chewing time. Error bars denote the standard deviation over a group of five volunteers, with

each volunteer having chewed the same gum twice for all time points. A. CFUs trapped per gum piece

obtained after molding, sonication and agar-plating. B. Total number of bacteria trapped per gum piece

obtained after dissolving the gum and performing qPCR.

Figure 3. Diversity of bacterial strains and species trapped in chewed gum in comparison with the

bacterial diversity in the salivary microbiome and the micobiome adhering to tooth surfaces. A. The

number of bands in DGGE gels in bacterial DNA obtained from pieces of chewed gum as a function of the

chewing time. Error bars denote the standard deviation over a group of five volunteers. No statistically

significant differences were observed. B. Percentage of species detected in the microbiome adhering to tooth

surfaces or in the salivary microbiome relative to the number of species found in chewed gum (10 min of

chewing) set at 100%. Error bars denote the standard deviation over a group of five volunteers. No

statistically significant differences were observed. C. Percentage of species found in chewed gum based on

origin, i.e. found in chewed gum and the adhering microbiome, chewed gum and the salivary microbiome and

found in gum and both microbiomes. The category “other origin” indicates species that were solely found in

chewed gum and below detection in the salivary and in the adhering microbiome.

Discussion

In this paper we provide evidence that bacteria are trapped inside gum pieces chewed by

human volunteers and therewith may contribute to the maintenance of oral health. The number

of bacteria trapped in chewed gums were determined using two distinctly different methods.

Finger-chewing and subsequent sonication and agar-plating demonstrated that

approximately 1–1.5 log-units less than the number of bacteria chewed-in could be retrieved,

regardless of the type of gum or bacterial strain involved, i.e. coccus- or rod-shaped microorganisms

(Fig. 1A). Although this recovery is confined to the surface layer of the gums amenable

to sonic removal of chewed-in bacteria and therefore relatively low, it allows to culture the bacteria

retrieved and express them in terms of CFUs. Compared to qPCR, which requires chemical

dissolution of the gum and bacterial lysis to determine the presence of genomic DNA from

bacteria trapped in chewed gums, agar-plating yields lower numbers of trapped bacteria, likely

because qPCR includes both dead and live bacteria [32] while agar-plating only reports viable

ones. Whereas agar plating yielded results that were independent of the bacterial strain involved,

Ct values obtained in qPCR were somewhat strain-dependent (Fig. 1B), possibly due to

differences in efficacy of lysis of the different strains and the relative efficiencies of the primer

pairs used. However, since calibration curves are applied to bacterial samples of unknown composition,

the small strain-dependent differences in Ct values were neglected and average calibration

curves were calculated and employed.

Both methods indicate a slow but significant decrease in bacterial trapping with increasing

chewing time in human volunteers after an initial maximum, regardless of the type of gum involved.

Whereas the initial gum bases are thus most adhesive to oral bacteria (Fig. 2) continued

chewing changes the structure of the gums, decreasing the hardness of the gum due to uptake

of salivary components [33] and release of water soluble components. This presumably affects

the adhesion of bacteria to the gum [34], causing a release of initially trapped, more weakly adhering

bacteria from the gum. Such a change in composition of trapped bacteria is supported

by the observation that the diversity of species trapped in chewed gum increases with chewing

time (Fig. 3A).

Despite an increasing diversity in species developing over time in chewed gums, there is a

gradual decrease in the number of bacteria trapped in chewed gum over time. This can be attributed

to a decrease in bacterial concentration in saliva during chewing, shown in earlier reports

[13]. However, alternative explanations exist as well, especially since this decrease is far

Figure 4. SEM visualization of bacteria trapped in a piece of chewed gum. Scanning electron

micrograph of a bacterium (indicated by white arrow) trapped in a chewed gum piece of gum A. The scale bar

indicates 1 μm. more prominent for the numbers of CFUs retrieved than for the total numbers of bacteria

found by qPCR in chewed gum. This difference in decrease suggests that bacteria are killed

during their entrapment in the gum by sweeteners like xylitol, food preservatives or flavoring

agents like spearmint and peppermint, which are reported to have antimicrobial properties

[9,35–37].

Numbers of bacteria trapped in a chewed piece of gum amount around 108 depending on

the time of chewing and retrieval method. Although this number may be considered low, it

shows that when gum is chewed on a daily basis, it may contribute on the long-term to reduce

the bacterial load in the oral cavity, which is supported by observations that long-term studies

on the use of chewing gum cause a reduction in the amount of oral biofilm [38]. Bacteria

trapped in chewed gum can originate either from the salivary microbiome or the adhering

microbiome on teeth, but also from the tongue, gums or oral mucosal surfaces from which we

did not sample. No DNA was detected in unchewed gum pieces. Saliva harbors up to 109 microorganisms

per ml before chewing [11,39]. Assuming a volume of saliva of around 1 ml in

the oral cavity, our results indicate that chewing of one piece of gum removes around 10% of

the oral microbial load in saliva. However, as our DGGE results pointed out, saliva does not

necessarily have to be the source of the bacteria found trapped in chewed gum. Making the alternative

assumption that all bacteria trapped in chewed gum come from the adhering microbiome,

we can place this number in further perspective by comparing it to the number of

bacteria removed by toothbrushing. Using a new, clean toothbrush without any toothpaste reportedly

removes around 108 CFUs per brush [39,40], which would put chewing of gum on par

with the mechanical action of a toothbrush. Moreover, also the mechanical action of floss wire

removes a comparable number of bacteria from the oral cavity than does chewing of a single

piece of gum, as we established in a simple pilot involving 3 human volunteers who used 5 cm

of floss wire (unpublished). Chewing however, does not necessarily remove bacteria from the

same sites of the dentition as does brushing or flossing, therefore its results may be noticeable

on a more long-term than those of brushing or flossing [7,19,41].

Our findings that chewing of gum removes bacteria from the oral cavity, may promote the

development of gum that selectively removes specific disease-related bacteria from the human

oral cavity, for instance by using porous type calcium carbonate [42]. It is known that the key

to oral health is a balanced and diverse composition of the oral microbiome, although the exact

composition of what is tentatively called “the oral microbiome at health” is not known. Removal

of specific pathogens however, is directly in line with the general notion arising in dentistry

that oral diseases develop when the oral microbiome shifts its composition into a less diverse

direction [43]. In this respect, a gradual removal of bacteria from the oral cavity through regular

removal of low numbers of pathogens by chewing gum is preferable to sudden ecological

shifts that can change the relationship between the oral microbiome and the host as another

potential cause of disease [43].

Acknowledgments

We would like to thank all volunteers for their cooperation in this study.

Author Contributions

Conceived and designed the experiments: SWW HCMDM AMHJB. Performed the experiments:

SWW AMS BBG. Analyzed the data: SWW HCMDM AMS BBG AM HJB. Contributed

reagents/materials/analysis tools: DM AM. Wrote the paper: SWW HCMDM AMHJB.

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