Anaerobic Bacteria
Anaerobic Bacteria
Anaerobic Bacteria
a r t i c l e i n f o a b s t r a c t
Article history: In this study, we aimed to identify the cultivatable oral anaerobic bacterial distribution in oral cavity
Received 28 March 2020 by MALDI-TOF Biotyper. The bacterial distribution of three groups, including subjects with/without peri-
Received in revised form odontal disease, two clusters of age (60 years as the cutoff), and before/after treatment, were investigated
12 September 2020
in this study. There were 38 participants recruited in this study, involving 18 subjects with moderate
Accepted 15 September 2020
to severe periodontal-infected patients and 20 healthy controls. Total number of 126 bacterial species
Available online 22 September 2020
were identified by MALDI-TOF MS. The relative abundance of Streptococcus gordonii and Streptococcus
intermedius in periodontal patients is higher than healthy controls indicating potential biomarkers for
Keywords:
oral microbiota
periodontal disease. Participants with periodontal disease were subdivided in to two clusters of age (60
MALDI-TOF Biotyper years as the cutoff), 11 and 7 participants were age <60 years and>60 years, respectively. Meanwhile, the
periodontal disease incidence of Streptococcus pneumoniae and Streptococcus oralis infection were higher in the subjects above
60 years old than below. Moreover, the bacterial distribution between pre-treatment and post-treatment
was similar indicating that basic treatment without the ability to redistribute the microbiota.
In summary, the cultivable oral anaerobic bacteria were identified by MALDI-TOF MS and the bacte-
rial distribution shifting was shown to be associated with the progress of periodontal disease to aging
and basic treatment. This study provided information for diagnosis and treatment guidelines for oral
healthcare.
© 2020 Elsevier B.V. All rights reserved.
1. Introduction no obvious symptoms at the early stage. Besides, the effects of the
non-surgical treatment for periodontal disease was not long-term
Ninety percent of the global population was suffering from peri- stability resulted in the relapse and the following disease progres-
odontal disease [1]. High prevalence of periodontal disease made sion [3]. In addition, the prevalence of serious periodontal disease
it a public health concern [2]. The reason for the extremely high increased with age. Hence, age was also one of the risk factors
prevalence of periodontal disease is that it is a silent illness with related to periodontal disease [2]. The incidence rate of periodon-
titis was constant in individuals who older than 60 years [4]. The
quality of elderly life was also impacted by the disability result from
∗ Corresponding author at: Institute of Bioinformatics and Structural Biology &
the periodontal disease, such as tooth loss, periodontal ligament
Department of Medical Science, National Tsing Hua University, No.101, Kuang-Fu
loss, and destruction of surrounding alveolar bone [5]. Furthermore,
Rd. Sec.2, Hsinchu, 30013, Taiwan. periodontal disease was found to correlate with multiple systemic
∗∗ Corresponding author at: SDGs Teaching and Research Headquarters, Tzu Chi diseases, such as diabetes, cardiovascular diseases, and rheumatoid
University, No. 701, Sec. 3 Jhongyand Rd., Hualien, 97004, Taiwan. arthritis [6]. The high prevalence, disabled life and the association
E-mail addresses: wccheng@gms.tcu.edu.tw (W.-C. Cheng),
hlchan@life.nthu.edu.tw (H.-L. Chan).
https://doi.org/10.1016/j.jpba.2020.113647
0731-7085/© 2020 Elsevier B.V. All rights reserved.
2 Y.-S. Wei, Y.-R. Chang, Y.-T. Tsai et al. / Journal of Pharmaceutical and Biomedical Analysis 192 (2021) 113647
of periodontal diseases with systemic diseases showed the dental guidelines and regulations. General inclusion criteria were as fol-
hygiene should be paid attention. lows: >20 years of age, no use of antibiotics and anti-inflammatory,
Periodontal disease is a bacteria-induced inflammatory disease immunosuppressive, antiepileptic, antihypertensive, or anticoagu-
caused by microbial dysbiosis [7]. The portion of gram-negative and lant drugs over the last 6 months. The exclusion criteria included
anaerobic organisms were high in subjects with periodontal dis- oral lesion, dental braces, denture, mouthwash allergy, pregnancy,
ease [8]. In addition, a recent systematic review has showed that use of birth control pills or serious systemic disease. Oral inclusion
periodontal disease probably was not caused by the presence of criteria were all subjects had at least 16 teeth and 6 front teeth
specific bacteria, such as Porphyromonas gingivalis and Tannerella at least. The subjects were subdivided into two following groups:
forsythia, but by dynamic changes of anaerobic bacterial distribu- patients with periodontal disease (n = 18) and healthy subjects (n
tion in the oral cavity [9]. Recent studies also showed the high = 20). The healthy subjects were selected without a diagnosis of
bacterial richness is related to poor periodontal conditions. Among periodontal disease and periodontal probing depth < 3 mm. The
them, Prevotella and Veillonella species ware associated with poor patients with periodontal disease were selected from patient with
oral status and old age. In addition, the specific pathogen changes a diagnosis of periodontal disease and periodontal probing depth>
were not significant after basic treatment (scaling and root plan- 3 mm. The patient groups underwent the first collection of samples
ning, SRP). The inability to effectively control microorganisms in after diagnosis and received basic treatment, scaling and root plan-
a long-term may be the reason for the short-term impact of SRP ning (SRP) for a month. SRP is a deep-cleaning method that uses
treatment [3,10]. Such findings also showed that the distribution manual instruments and/or ultrasonic scalers to remove plaque
of microorganisms in saliva can reflect oral health [11]. From the from the tooth surface underneath the gum line and from the root
research above, there is still the room of discussion on the dynamic surface. This nonsurgical periodontal therapy drastically reduces
changes of oral microorganisms and diseases [9,12]. inflammation induced by periodontal disease [21–23]. Afterwards,
Traditionally, most of results for identifying bacteria in oral the samples were collected again (second collection).
cavity were showed by detecting specific target bacteria but lost
the whole bacterial distribution information [13]. Amplification 2.2. Sample collection
of conserved regions of the 16S ribosomal RNA (rRNA) gene by
Gingival sulcus and saliva samples were collected from all
polymerase chain reaction (PCR) was used to identify microor-
subjects. Gingival sulcus samples from subjects with periodontal
ganisms currently. However, it is difficult to distinguish live and
disease and healthy subjects were collected by sterilized paper
dead bacteria and mislead quantitative results, expensive and
points, which were inserted into gingival sulcus for 10 seconds
time-consuming [13]. Matrix-assisted laser desorption ionization
each. For saliva collection, subjects were asked to spit whole saliva
time-of-flight mass spectrometry (MALDI-TOF MS) for microbial
into a 15-mL sterilized tube. Meanwhile, each sample was pre-
identification has revolutionized clinical laboratory practice, owing
served to a CMPTM Anaerobic TranSwab container before sample
to its low cost, fast identification speed, lower sample volumes and
preparation prior to bacterial culture.
high accuracy in comparison to classical laboratory procedures for
After removal from the CMPTM Anaerobic TranSwab, all samples
microbial identification, such as phenotypic tests involving bacte-
were processed within 10–20 minutes. A sample was transferred
rial culture, gram-staining, biochemical profiling, whole-genome
into 0.5 mL of Tryptic soy broth (TSB), and mixed with 0.5 ml of
DNA probes, and DNA–DNA hybridization [14]. MALDI-TOF MS
glycerol, then stored at −80 ◦ C. After finishing samples collection,
technique has been widely utilized in variety of field studies for
several samples were pooled into one. Next, 10,000× to 200,000×
identifying microbe, such as those diagnosing pathogens in uri-
dilutions (beginning from 10—5 to 2 × 10−6 ) of the saliva samples
nary tract, gut, oral and blood [14–16]. MALDI-TOF MS can be used
and 2× to 320× dilutions of the gingival sulcus samples were pre-
to identify bacteria without any knowledge of the microbial tax-
pared. The diluted samples (100 L) were then transferred onto
onomic affiliation [16]. The microbial identification is based on
prereduced Anaerobic Blood Agar (TSB with 5% defibrinated sheep
scoring algorithms to match analyzed spectra with reference spec-
blood, 0.5% yeast extract, 0.05% cysteine HCl-H2 O, 0.5 mg/ml hemin
tra of peptides and proteins extracted from microorganisms of
and 2 g/ml vitamin K1) and chocolate agar for anaerobic incuba-
interest in databases which renewed accompanying by the research
tion (anaerobic gas mixture, 80% N2 , 10% CO2 , 10% H2 , 37 ◦ C, 4 days’
continuously [17]. Further, the studies have demonstrated the
duration) in a Whitley DG250 Workstation.
accuracy of the MALDI-Biotyper to genus-level identification rate
of 95.2% and a species-level rate of 82.5% [18–20]. Thus, MALDI-TOF 2.3. Microbial identification and quantification: Identification by
MS has its advantage in species-level identification [13]. MALDI-TOF MS analysis
In order to provide information on the distribution of anaerobic
microbiota for supporting diagnosis and treatment in the future. Bacterial colonies were transferred to a target polished steel
The dynamic changes of oral bacteria were investigated in this plate (MBT 384, Bruker Daltonics Inc.). The proteins of the samples
study. The aim of the study is to investigate the bacterial distri- were extracted with 1 L 70% formic acid (Sigma). After air-drying,
bution in oral cavity by identifying bacteria by MALDI-TOF MS, and they were overlaid with 1 L of a matrix solution (10 mg/ml solu-
compared the difference in each groups with/without periodontal tion of ␣-cyano-4-hydroxycinnamic acid (HCCA), in a mixture of
disease, two clusters of age (60 years as the cutoff) and before/after 50% acetonitrile, 47.5% ultra-pure water, and 2.5% trifluoroacetic
treatment. acid). After repeated the air-drying process, the dried samples were
analyzed on an Autoflex III MALDI-TOF mass spectrometer (Bruker
Daltonik GmbH, Leipzig, Germany). The spectra were recorded in
2. Materials and methods
the range of 2,000–20,000 Da at the maximal laser frequency.
2.1. Settings and Participants Raw spectra were then automatically analyzed by means of the
MALDI Biotyper 3.1 software package (default settings; Bruker Dal-
The subjects in the study were enrolled from a single institution: tonik GmbH, Bremen, Germany, Biotyper® database renewed at
Taipei Medical University in Taipei, Taiwan. The protocol approval 2014/9/8). Approximately 5989 species can be detected by this
(No: NCT03345329, November 17, 2017) was obtained from Taipei technology. Obtained log scores ranging from 0 to 3.00 which
Medical University-Joint Institutional Review Board at August 24th, according to the criteria recommended by the manufacturer. Scores
2015, and informed written consent was obtained from all sub- ≥ 1.7 as a confidence identification and scores <1.7 as no reliable
jects. All methods were performed in accordance with the relevant identification.
Y.-S. Wei, Y.-R. Chang, Y.-T. Tsai et al. / Journal of Pharmaceutical and Biomedical Analysis 192 (2021) 113647 3
Fig. 1. Distributions of bacteria were analyzed by means of MALDI Biotyper in saliva samples collected from subjects with or without periodontal disease and subjects with
periodontal disease after treatment. A heatmap representing the relative abundance of bacteria in the saliva that were cultured in an anaerobic environment and identified
using MALDI-TOF MS in this study. Subjects were classified into three groups: H means samples from periodontal healthy subjects; P denotes samples from patients with
periodontal disease; TX means samples collected from subjects with periodontal disease after SRP. S means relative abundance within a bacterial distribution in saliva
according to MALDI Biotyper results.
Relative abundance (RA): Percentages were calculated by the Statistics were performed using GraphPad Software. Data
detectable bacterial colonies count of each species by the total shown are the means and standard errors of the means (SEM).
detectable bacterial colonies count. Significances were established by the Student’s t test.
4 Y.-S. Wei, Y.-R. Chang, Y.-T. Tsai et al. / Journal of Pharmaceutical and Biomedical Analysis 192 (2021) 113647
Fig. 2. Distributions of bacteria identified by MALDI-TOF MS were analyzed in gingival sulcus samples collected from subjects with or without periodontal disease and subjects
with periodontal disease after treatment. A heatmap representing the relative abundance of bacteria in the gingival sulcus that were cultured in an anaerobic environment
and identified using MALDI-TOF MS in this study. The subjects were classified into three groups: H means samples from periodontal healthy subjects; P indicates samples from
patients with periodontal disease; TX denotes samples collected from subjects with periodontal disease after SRP. G means relative abundance within a bacterial distribution
in gingival sulcus according to MALDI Biotyper results.
Fig. 3. The relative abundance levels of bacteria in saliva collected from subjects with or without periodontal disease and subjects with periodontal disease after treatment.
(a) Streptococcus pneumoniae and (b) Streptococcus salivarius show changes of relative abundance in saliva from subjects with periodontal disease versus healthy subjects
and subjects with periodontal disease after versus before treatment. (* P < 0.05, **P < 0.01, ***P < 0.001 according to the unpaired t test.)
Fig. 4. The relative abundance levels of bacteria in gingival sulcus samples collected from subjects with or without periodontal disease and subjects with periodontal disease
after treatment. (a) Streptococcus gordonii, (b) Streptococcus intermedius, (c) Actinomyces graevenitzii, (d) Actinomyces odontolyticus, (e) Fusobacterium periodonticum, (f) Gemella
sanguinis, (g) Streptococcus parasanguinis, (h) Streptococcus pneumoniae, and (i) Streptococcus salivarius show changes of relative abundance in gingival sulcus samples from
subjects with periodontal disease versus healthy subjects and subjects with periodontal disease after versus before treatment. (* P < 0.05, **P < 0.01, ***P < 0.001 according
to the unpaired t test.)
6 Y.-S. Wei, Y.-R. Chang, Y.-T. Tsai et al. / Journal of Pharmaceutical and Biomedical Analysis 192 (2021) 113647
and received basic treatment, scaling and root planning (SRP) for The relative abundance of S. parasanguinis in the saliva from the
a month then collection again. To investigate the differences in subjects older than 60 years old was significantly higher than that
the anaerobic bacterial oral flora of participants with/without peri- in subjects younger than 60 years of age (P = 0.0147, Fig. 5). Signif-
odontal disease, two clusters of age (60 years as the cutoff [25]) and icant differences were also found by Student’s t test in the relative
before/after treatment, the bacteria cultured in the anaerobic envi- abundance of the 13 species in gingival sulcus (Figs. 6 and 7). The
Fig. 6. Distributions of bacteria found in gingival sulcus samples (collected from subjects with periodontal disease younger or older than 60 years and those subjects after
treatment) were identified by means of MALDI Biotyper. A heatmap representing the relative abundance of bacteria in gingival sulcus that were cultured in an anaerobic
environment and were identified using MALDI-TOF MS in this study. The samples were classified into four groups: “<60” means periodontitis patients, under 60 years of age;
“>60” denotes periodontitis patients, above 60 years of age; “TX” indicates the samples collected from periodontitis patients after basic treatment.
Y.-S. Wei, Y.-R. Chang, Y.-T. Tsai et al. / Journal of Pharmaceutical and Biomedical Analysis 192 (2021) 113647 7
Fig. 7. MALDI Biotyper results on bacterial distributions in gingival sulcus collected from subjects with periodontal disease younger or older than 60 years and those subjects
after treatment. (a) Veillonella parvula, (b) Actinomyces oris, (c) Streptococcus sanguinis, (d) Streptococcus constellatus, (e) Staphylococcus epidermidis, (f) Campylobacter concisus,
(g) Gemella morbillorum, (h) Streptococcus pneumoniae, (i) Streptococcus oralis, (j) Fusobacterium nucleatum, (k) Aggregatibacter aphrophilus and (l) Campylobacter rectus. The
bacterial distributions in gingival sulcus (G) are shown in the bar plot. The samples were subdivided into four groups: under 60 years, periodontitis (<60), and above 60 years,
periodontitis (>60), with or without basic treatment (TX). Gingival sulcus sample was collected by sterilized paper points and put into the 0.5 ml TSB, and a half was stored
with glycerol at —80 ◦ C. (*P < 0.05, **P < 0.01, ***P < 0.001 according to the unpaired t test.)
8 Y.-S. Wei, Y.-R. Chang, Y.-T. Tsai et al. / Journal of Pharmaceutical and Biomedical Analysis 192 (2021) 113647
relative abundance of five out of the 13 species in gingival sulcus In addition, S. gordonii can enter the blood when there is a wound
was comparatively higher in subjects under 60 years of age than in the oral cavity and cause endocarditis, leading to dysfunction
in subjects over 60 years of age. Veillonella parvula abundance was of the heart valves [29,30]. S. intermedius is normally found in the
eight-fold higher (P = 0.0029), Actinomyces oris abundance was five- oral cavity, but can also be present in the upper respiratory tract
fold higher (P = 0.0057), and Streptococcus sanguinis abundance was and human feces [31]. The membrane of S. intermedius contains sur-
four-fold higher (P = 0.0304; Fig. 7a–c). In addition, Staphylococcus face protein Antigen I/II, which can assist bacterial attachment, and
epidermidis and Streptococcus constellatus were found only in gingi- S. intermedius secretes the cytotoxin intermedilysin. The latter can
val sulcus of subjects under 60 years old (P = 0.0236 and P = 0.0309, cause lysis of human cells and increase the risk of deep-seated puru-
respectively; Fig. 7d and 7e). The relative abundance of eight species lent abscesses [32,33]. The biofilm created by S. intermedius can
in gingival sulcus was comparatively higher in subjects over 60 also protect itself from antibiotics and the host’s immune response
years of age than in subjects younger than under 60 years of [34]. In summary, our data showed that S. pneumoniae in saliva,
age. Campylobacter concisus abundance was seven-fold higher (P S. gordonii and S. intermedius in gingival sulcus were not report to
= 0.0332), Gemella morbillorum abundance was 27-fold higher (P = correlate with periodontal disease directly, but the relative abun-
0.0073), S. pneumoniae abundance was 10-fold higher (P = 0.0139), dance of this species were higher in samples from subjects with
Streptococcus oralis abundance was six-fold higher (P = 0.0192), periodontal disease than healthy. The results indicate that S. pneu-
and Fusobacterium nucleatum abundance was 11-fold higher (P = moniae, S. gordonii and S. intermedius might be the indicator for oral
0.0013; Fig. 7f–j). In addition, Aggregatibacter aphrophilus, Campy- health.
lobacter rectus, were found only in gingival sulcus samples from After investigating the correlation between bacterial distri-
subjects over 60 years old (P = 0.0440, P = 0.0255, and P = 0.0032, bution and periodontal disease, the subjects with periodontal
respectively; Fig. 7k and 7 l). disease were received basic treatments and samples would collect
after treatment to investigate the change of bacterial distribution
before/after basic treatment. According to Student’s t test, basic
3.4. Identification of bacterial samples by MALDI-TOF MS before
treatment significantly decreased the relative abundance of certain
and after basic treatment
bacterial species, such as S. pneumoniae found in saliva. Neverthe-
less, in the gingival sulcus samples, the relative abundance levels
The relative abundance of S. pneumoniae in the saliva from the
of A. graevenitzii, A. odontolyticus, S. parasanguinis, and S. salivar-
subjects after treatment decreased two-fold in comparison with
ius were found to be higher after basic treatment. Among them,
pretreatment samples (P = 0.0086; Fig. 3a). In a parallel experi-
Actinobacteria sp. are were common cause of infection in dental
ment, the relative abundance of four species in gingival sulcus, A.
procedures and oral abscesses [35]. The results indicate that basic
graevenitzii, A. odontolyticus, S. parasanguinis, and S. salivarius, was
treatment is insufficient for bacterial control or redistribution.
found to be significantly higher in subjects post-treatment than in
Even though our data showed that the dynamics of bacterial dis-
subjects before basic treatment (P < 0.05; Fig. 4c, d, g, i).
tribution in different disease condition, age, and treatment, there
are still some limitation. Three limitations in this study were the
4. Discussion coverage of database, bacterial culture condition, and the effects
of environmental factors on subjects. First, 70% of all the bacterial
Periodontal disease has been reported as a bacteria-induced isolates were analyzed, implying that 30% of bacterial isolates from
inflammatory disease [2,7]. Even though the specific bacteria, such our samples could not be identified. Moreover, two species Tan-
as Porphyromonas gingivalis and Tannerella forsythia were shown nerella forsythia and Treponema denticola, which the most closely
the association to periodontal diseases [9]; however, the large associated with severe periodontitis in Scoransky’s studies, were
scale thoroughly discover periodontal disease-associated bacterial not involved in the database. This is likely because identification of
is required for periodontal therapy. To investigate the dynamic certain species had not been programmed in the MALDI Biotyper 3.1
changes of anaerobic bacterial distribution associated to periodon- software package. The results indicate that the MALDI Biotyper 3.1
tal disease, samples were cultured in the anaerobic environment software developers should expand its database to cover a wider
for 4 days and identified by MALDI-TOF MS to investigate the dif- range of bacterial strains to identify strains that may be disease-
ference of bacterial distribution between subjects with/without associated. Second, Anaerobic Blood Agar and chocolate agar, which
periodontal disease. Briefly, total of 126 species were identified was used to cultured bacteria in this study, could not culture Tan-
by MALDI-TOF MS in saliva and gingival sulcus samples from 38 nerella forsythia (which needs N-acetylmuramic acid for growth)
subjects. The relative abundance levels of S. pneumoniae and S. sali- and Treponema denticola (which needs spirochete medium). Third,
varius were significantly different between the saliva of healthy environmental factors, such as smoking and antibiotic use, have a
subjects and patients with periodontal disease in this study. S. significant influence on the bacterial distribution, should be con-
pneumoniae normally resides without active infection in the upper sidered in the future. Therefore, amplification of the database,
respiratory tract and can cause pneumonia and meningitis when increasing of culture conditions, consideration of the impact of
immunity is compromised [26]. However, the relative abundance environmental impact factors on the subject will lead to a more
of S. salivarius in saliva was found to be higher in healthy subjects complete oral bacterial distribution.
than in patients with periodontal disease. S. salivarius is found in In conclusion, we identified 126 species and the dynamics of
the normal bacterial community of the oral cavity, can secrete an bacterial distribution showed in our study. Given the changes of
antibiotic peptide, BLIS (bacteriocin like inhibitory substance), to bacterial distribution are related to the oral health. Meanwhile,
prevent oral infections, and can serve as a probiotic [27]. There- our data showed that the bacterial distribution is correlated to
fore, our results imply that the maintenance of S. salivarius directly the elderly. The incidence of S. pneumoniae and S. oralis infection
correlates with the host’s periodontal health. rate were found higher in the subjects above 60 years old. More-
The relative abundance of S. gordonii and S. intermedius in gingi- over, the data of bacterial distribution between pre-treatment and
val sulcus was higher in patients with periodontal disease than in post-treatment indicating that basic treatment is insufficient. In
healthy subjects. Previous study showed that S. gordonii is involved summary, we have identified anaerobic bacteria by MALDI-TOF Bio-
in the formation of a biofilm known as tooth plaque; although S. typer system and provided information for diagnosis and treatment
gordonii is not a direct pathogen, the plaque formation is one of the guidelines in the future.
contributing factors to periodontal disease and dental caries [28].
Y.-S. Wei, Y.-R. Chang, Y.-T. Tsai et al. / Journal of Pharmaceutical and Biomedical Analysis 192 (2021) 113647 9
Author Contributions Statement [12] Y. Zhang, et al., Human oral microbiota and its modulation for oral health,
Biomed Pharmacother 99 (2018) 883–893.
[13] H.C. Yeh, et al., Identification of microbiota in peri-implantitis pockets by
Hong-Lin Chan and Wen-Chi Cheng designed the research. Yu- matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,
Shan Wei wrote the manuscript text and prepared all figures. Sci Rep 9 (1) (2019) 774.
Yu-Shan Wei, Yi-Ting Tsai, Yi-Ting Yang, Shang-Hui Weng, and Lin- [14] K.S. Jang, Y.H. Kim, Rapid and robust MALDI-TOF MS techniques for microbial
identification: a brief overview of their diverse applications, J Microbiol 56 (4)
Fang Tseng carried out the experiment. Yi-Ru Chang, En-Chi Liao, (2018) 209–216.
Hsin-Yi Chen and Guan-Yu Lin make substantial contributions to [15] B. Samb-Ba, et al., MALDI-TOF Identification of the Human Gut Microbiome in
the acquisition of data. Hsiu-Chuan Chou and Alice Tinyu Hu super- People with and without Diarrhea in Senegal, PLOS ONE 9 (5) (2014) e87419.
[16] Y.-S. Wei, et al., Identification of hyperglycemia-associated microbiota
vised the project. All authors reviewed the manuscript.
alterations in saliva and gingival sulcus, Archives of Biochemistry and
Biophysics 682 (2020) 108278.
Funding [17] M.M. Bhatti, et al., Rapid identification of positive blood cultures by
matrix-assisted laser desorption ionization-time of flight mass spectrometry
using prewarmed agar plates, J Clin Microbiol 52 (12) (2014) 4334–4338.
This work was supported by MOST106-2311-B-007-002, and [18] P. Decristophoris, et al., Identification of Staphylococcus intermedius Group by
MOST 109-2327-B-400-001 grants (from the Ministry of Science MALDI-TOF MS, Syst Appl Microbiol 34 (1) (2011) 45–51.
[19] B. Schulthess, et al., Use of the Bruker MALDI Biotyper for identification of
and Technology, Taiwan) and R&D Piloting Cooperation Projects
molds in the clinical mycology laboratory, J Clin Microbiol 52 (8) (2014)
between Industries and Academia104A19 and 105A24 (from the 2797–2803.
Hsinchu Science Park, Taiwan) [20] B. Schulthess, et al., Evaluation of the Bruker MALDI Biotyper for identification
of Gram-positive rods: development of a diagnostic algorithm for the clinical
laboratory, J Clin Microbiol 52 (4) (2014) 1089–1097.
Declaration of Competing Interest [21] J. Carnio, et al., Nonsurgical periodontal therapy to treat a case of severe
periodontitis: A 12-year follow-up, J Am Dent Assoc 146 (8) (2015) 631–637.
The authors report no declarations of interest. [22] I. Sanz, et al., Nonsurgical treatment of periodontitis, J Evid Based Dent Pract
12 (3 Suppl) (2012) 76–86.
[23] J. Liu, et al., Clinical and microbiologic effect of nonsurgical periodontal
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nurses. Additionally, the authors would like to thank Amber Kao for [25] P.E. Petersen, T. Yamamoto, Improving the oral health of older people: the
proofreading and editing of the manuscript. approach of the WHO Global Oral Health Programme, Community Dentistry
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