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ORAL MICROBIOLOGY                                                                                                                                   www.stomaeduj.com




PREVOTELLA SPECIES – THE MOST PREVALENT




                                                                                                                                                  Original Articles
BLACK-PIGMENTED ANAEROBIC BACTERIA AMONG
OROPHARYNGEAL ISOLATES OBTAINED FROM
A SAMPLE OF HEALTHY YOUNG ADULTS
Gabriela Băncescu1a* , Lidia Sfetcu1b , Andreea Cristiana Didilescu2c , Adrian Anton Băncescu3d
1
 Department of Microbiology, Faculty of Dental Medicine, "Carol Davila" University of Medicine and Pharmacy Bucharest, Bucharest, Romania
2
 Department of Embryology, Faculty of Dental Medicine, "Carol Davila" University of Medicine and Pharmacy Bucharest, Bucharest, Romania
3
 Department of Epidemiology, Faculty of Medicine, "Carol Davila" University of Medicine and Pharmacy Bucharest, Bucharest, Romania

a
  MD, MSc, PhD, Professor, Head; e-mail: gabi.bancescu@yahoo.com; ORCIDiD: https://orcid.org/0000-0003-2827-2465
b
  DMD, PhD, Assistant Professor; e-mail: lidia.ciobanu@ymail.com; ORCIDiD: https://orcid.org/0000-0002-4023-5053
c
 DMD, MPhil, PhD, Professor, Head, Vice-Dean; e-mail: andreea.didilescu@gmail.com; ORCIDiD: http://orcid.org/0000-0002-2915-5124
d
  MD, MSc, PhD, Associate Professor; e-mail: adrianbancescu@yahoo.com; ORCIDiD: https://orcid.org/0000-0003-4164-9810

ABSTRACT                                                                             https://doi.org/10.25241/stomaeduj.2020.7(2).art.1

Introduction The black-pigmented Prevotella - Porphyromonas group comprises members of the normal
flora of the oral cavity, oropharyngeal, intestinal and genitourinary tract, but may be associated with various
infections too. The purpose of this study was to identify the species of this anaerobic group which frequently
colonize the oropharynx in clinically healthy young adults.
Methodology The microbiological investigation was carried out on a strain collection of 93 dark-pigmented
anaerobic isolates originated from the oropharynx of healthy dental students, at the Department of
Microbiology, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy - Bucharest.
All isolates of Gram-negative bacilli were identified at the genus and species level by conventional methods,
MASTRING ID (MAST Group Ltd., U.K.) and Rapid ID 32 A system (BioMérieux, France), while the isolates of
anaerobic cocci were tested only by MASTRING ID.
Results The microscopy of the Gram-stained smears and the results of the MASTRING ID test performed
with the 93 black-pigmented anaerobic isolates indicated that 77 strains were Gram-negative bacilli and/
or coccobacilli, while 16 strains were Gram-positive cocci. The identification of the Gram-negative bacilli
at the species level concluded that 57 strains belonged to Prevotella denticola, 18 strains to Prevotella
melaninogenica and 2 strains to Prevotella intermedia.
Conclusion P. denticola and P. melaninogenica might be considered the main species of the black-pigmented
Prevotella-Porphyromonas group which colonize the oropharynx in healthy young adults. These species are
usually beta-lactamase producers and their high rate of oropharyngeal colonization should be considered
when antibiotics are needed in oral infections therapy.
KEYWORDS
Oropharynx, Microbiota, Anaerobes, Black-Pigmented Bacteria, Prevotella.

1. INTRODUCTION                                                                secretion. The oropharynx microbiota is complex
                                                                               and comprises hundreds of microbial species [2,3],
The oropharynx comprises the palatine tonsils,                                 most of them organized in biofilms associated
tongue base, soft palate and posterior pharyngeal                              with the respective microenvironment [4]. About
wall and is lined by a nonkeratinized stratified                               80% of the local normal flora is represented by the
squamous epithelium [1]. The oropharynx mucosa                                 viridans streptococci and commensal species of
comes in contact with saliva and nasopharyngeal                                Haemophilus and Neisseria [5,6]. Besides different



               OPEN ACCESS This is an Open Access article under the CC BY-NC 4.0 license.
               Peer-Reviewed Article
    Citation: Băncescu G, Sfetcu L, Didilescu AC, Băncescu AA. Prevotella species – the most prevalent black-pigmented anaerobic bacteria among
    oropharyngeal isolates obtained from a sample of healthy young adults. Stoma Edu J. 2020;7(2):87-93.
    Received: March 19, 2020; Revised: April 17, 2020; Accepted: May 20, 2020; Published: May 22, 2020
    *Corresponding author: Professor Gabriela Băncescu, MD, MSc, PhD, Department of Microbiology, Faculty of Dental Medicine, "Carol Davila"
    University of Medicine and Pharmacy (CDUMPB), D. Gerota Str. 19-21, RO-020032-Bucharest, Romania.
    Tel./Fax: +40 771027131; e-mail: gabi.bancescu@yahoo.com
    Copyright: © 2020 the Editorial Council for the Stomatology Edu Journal.




Stoma Edu J. 2020;7(2): 87-93                                                                      pISSN 2360-2406; eISSN 2502-0285                  87
                     Băncescu G, et al.
www.stomaeduj.com



                     species of Mycoplasma, Corynebacterium and                in human pathology [23]. The present study intended
 Original Articles   staphylococci, the oropharynx may harbor also             to contribue to the investigation of black-pigmented
                     pathogenic or potentially pathogenic bacteria like:       Gram-negative anaerobic bacilli, since the anaerobic
                     Streptococcus pyogenes, Streptococcus agalactiae,         bacteria are not commonly cultivated by many
                     Streptococcus dysgalactiae subsp. equisimilis, S.         microbiology laboratories. Thus, the aim of this
                     pneumoniae, Neisseria meningitidis, Haemophilus           study was to identify the species belonging to this
                     influenzae or Moraxella catarrhalis [6-14]. Some          bacterial category which colonize the oropharynx in
                     studies reported that the dominant bacteria of            healthy young adults most frequently.
                     the normal oropharyngeal flora are the anaerobes
                     [15]. The persistence of anaerobic bacteria is            2. METHODOLOGY
                     favored by the anaerobic conditions found into the
                     depth of the biofilm covering the oropharyngeal           The microbiological investigation was performed
                     mucosa, mainly due to the oxygen consumption              on a collection of 93 black-pigmented anaerobic
                     by aerobic and facultative anaerobic bacteria. The        bacterial strains stored at -700C, at the laboratory of
                     mucosal convolutions and especially the tonsils           the Department of Microbiology, Faculty of Dental
                     crypts may be considered important anaerobic              Medicine, “Carol Davila” University of Medicine
                     microhabitats. At these sites, the strictly anaerobes     and Pharmacy (CDUMP) Bucharest. The respective
                     are found in high proportion with Prevotella being        strains originated from oropharyngeal swab samples
                     the most frequently isolated species [16,17]. Most        collected from 29 male dental students and 64
                     Gram-negative anaerobic bacteria colonizing the           female dental students, aged 20-34 years, at the
                     oropharynx in healthy adults belong to the following      same Microbiology department, in April 2018. They
                     genera: Prevotella, Fusobacterium, Leptotrichia           were second-year students, were clinically healthy
                     and Veillonella [2,18-22]. Some researchers also          and had not taken antibiotics in the last 6 months.
                     found Porphyromonas at this site, but except for          Twenty-four of them were smokers: 7 male subjects
                     Prevotella spp., the other anaerobes were isolated        and 17 female subjects. Prior to the present study,
                     in much smaller percentages than the facultative          cultures from the 93 oropharyngeal swab samples
                     anaerobes, such as: streptococci, staphylococci           were performed on Schaedler agar with 5% sheep
                     and diphteromorphs [16]. The conclusion of a              blood (BioMérieux, France), which were incubated
                     Norwegian-American research team that applied             in GENbox, with GENbox anaer sachet and Anaer
                     cultural-independent molecular techniques to              indicator (BioMérieux, France), at 350C, for 10
                     determine the microbial diversity of the oral flora       days, with examination every 48h. All isolates that
                     was that there is a distinctive dominant oral flora       developed black pigment and showed negative
                     in healthy subjects, with site-specificity and high       results in the aerotolerance test were stored in
                     diversity [23]. The same team found an unexpected         cryobiles, at -700C and constituted the collection of
                     high diversity at tonsils level (more than 50 different   strains used in the present study. At the beginning
                     species) with high variation between subjects [23].       of this study conducted in the second semester
                     Thus, Prevotella and Porphyromonas spp. were              of 2018, the black-pigmented anaerobic strains
                     isolated from some subjects, but were missing             belonging to the above mentioned collection were
                     in others who were harboring mostly bacteria              checked for their morphotinctorial characteristics by
                     belonging to the phylum Firmicutes [23].                  microscopic examination of Gram-stained smear. All
                     Some bacterial community analysis indicated               Gram-negative bacilli strains were further identified
                     that the main group associated with the throat            by the conventional methods, MAST ID MID8
                     microbiota comprises: species belonging to                ANAEROBE ID RING/MASTRING ID (MAST Group Ltd.,
                     Streptococcus and other genera of Firmicutes,             U.K.) and Rapid ID 32 A system (BioMérieux, France).
                     species belonging to the family of Pasteurellaceae,       The presumptive identification of the Gram-negative
                     Fusobacterium spp. and Actinomyces spp., while the        bacilli strains was performed by testing their ability to
                     main group associated with the tonsils includes           grow in the presence of 20% bile (by streaking them
                     species of Streptococcus, Mogibacterium and other         on Bacteroides bile-esculin agar, BBE) and by testing
                     Firmicutes, Fusobacterium spp., Prevotella spp. and       their susceptibility to: vancomycin (5 μg), kanamycin
                     members of the family Pasteurellaceae [24]. In healthy    (1000 μg), colistin sulphate (10 μg), erythromycin
                     persons, the core microbiome plays a major role           (60 μg), penicillin G (2 units) and rifampicin (15 μg)
                     in homeostasis [25]. In children, an oropharyngeal        by applying the MASTRING ID on blood agar plates
                     microbiome similar to that of adults was described,       (BioMérieux, France) seeded with bacterial inoculum
                     but richer in Prevotella, Neisseria, Granulicatella,      adjusted to the turbidity of 2 McFarland standard.
                     Porphyromonas and Fusobacterium [26]. At present          The plates were incubated in anaerobic atmosphere,
                     high efforts are required for advanced research           at 350C, for 48h. Bacteroides fragilis ATCC 25285 was
                     of normal flora of the oral cavity and oropharynx,        used as quality control. These tests were interpreted
                     estimating that future findings may substantially         based on the indication mentioned in the textbooks
                     contribute to understanding the role played by the        of diagnostic microbiology [27], completed with
                     microorganisms with oral or oropharyngeal habitat         the recommendation given by the MASTRING ID



    88               Stoma Edu J. 2020;7(2): 87-93                                            pISSN 2360-2406; eISSN 2502-0285
Black-pigmented anaerobes that colonize the oropharynx
                                                                                                                                               www.stomaeduj.com




                                                                                                                                             Original Articles
 Figure 1. MASTRING ID test applied on a strain of black-pigmented         Figure 2. The distribution of the 77 strains of black-pigmented
Gram-negative anaerobic bacilli.                                          Gram-negative anaerobic bacilli by species.


                                                                          found susceptible to penicillin. The interpretation
                                                                          of the tests according to the textbook of diagnostic
                                                                          microbiology [27] and MASTRING ID producer
                                                                          indicated that all these bacilli strains belonged to the
                                                                          genus Prevotella. The Rapid ID 32 A system indicated
                                                                          that 57 strains were Prevotella denticola, 18 strains
                                                                          were Prevotella melaninogenica and 2 strains were
                                                                          Prevotella intermedia (Fig. 2). In 5 subjects, pairs of P.
                                                                          denticola - P. melaninogenica were isolated. Twenty
                                                                          of the 72 students with black-pigmented Prevotella
                                                                          oropharyngeal colonization were smokers. Figure 3
 Figure 3. P. intermedia strain identified by the Rapid ID 32 A system.
                                                                          presents the biochemical profile of a P. intermedia
                                                                          strain obtained with the Rapid ID 32 A gallery (Fig. 3).
                                                                          A p-value of 0.5737 was found when applying the
producer. In addition, the MASTRING ID test was also                      Fisher exact test, indicating no statistically significant
applied to the coccus-shaped anaerobic isolates.                          correlation between smoking and colonization
The Fisher exact test was used to find any statistically                  of the oropharynx with black-pigmented Gram-
significant association between smoking (data on                          negative anaerobic bacilli.
this habit being received from students prior to this
study, when oropharyngeal swab samples were                               4. DISCUSSION
collected) and oropharynx colonization with black-
pigmented Gram-negative anaerobic bacilli species.                        The normal oropharyngeal flora may show variability
The chosen significance level was p ≤ 0.05.                               among healthy individuals due to external factors
                                                                          and ecological relationship. The investigation of the
3. RESULTS                                                                microbial relationship may contribute to clarifying
                                                                          many underlying aspects [28]. Anaerobic bacteria
The microscopic examination of the Gram-stained                           are not currently investigated by many laboratories
smears from the culture of dark-pigmented                                 due to the laborious and expensive work required.
anaerobic strains indicated that 77 of the 93 isolates                    Porphyromonas and Prevotella (previously belonging
were Gram-negative bacilli and/or coccobacilli. The                       to the genus Bacteroides) also include, besides
microscopy showed that the other 16 strains were                          non-pigmented species, dark-pigmented species
Gram-positive cocci (arranged mainly in irregular                         like: Porphyromonas gingivalis, Porphyromonas
clusters) and this was in complete agreement with                         endodontalis, Porphyromonas asaccharolytica, and
the results of the MASTRING ID. Figure 1 illustrates                      Prevotella intermedia, Prevotella nigrescens, Prevotella
the result of the MASTRING ID test applied to a                           melaninogenica, Prevotella loescheii, Prevotella
strain of black-pigmented Gram-negative anaerobic                         denticola and Prevotella corporis, respectively
bacilli (Fig. 1). The microbiological investigation                       [29]. This study focused on the investigation of
carried out on the dark-pigmented Gram-negative                           the oropharynx colonization by dark-pigmented
anaerobic bacilli and/or coccobacilli isolates                            Prevotella and Porphyromonas species. The culture
showed that all of them were susceptible to 20%                           method allowed the isolation of the black-pigmented
bile, rifampicin and erythromycin, but resistant to                       anaerobic strains. Because the development of
kanamycin and vancomycin. The results concerning                          dark-pigment is usually a delayed process, primary
the susceptibility to colistin and penicillin G varied,                   cultures (which were obtained prior to this study)
45 strains being found susceptible and 32 strains                         were checked throughout the incubation period.
resistant to colistin, while only 19 strains were                         The present study began with the selection of



Stoma Edu J. 2020;7(2): 87-93                                                                 pISSN 2360-2406; eISSN 2502-0285                  89
                     Băncescu G, et al.
www.stomaeduj.com



                     the bacilli strains among the 93 black-pigmented            greater number of species belonging to the genera:
 Original Articles   anaerobic isolates and continued with the species           Megasphaera, Streptococcus, Veillonella, Atopobium,
                     identification, based on both conventional and              Eggerthella, Dorea, Anaerovorax, Eubacterium and
                     rapid methods. P. denticola, P. melaninogenica and P.       to the family Erysipelotrichaceae [24]. Many of these
                     intermedia were the only black-pigmented anaerobic          bacteria can also be involved in oral infections.
                     bacilli species isolated from the oropharynx in this        Some researchers found that significant changes
                     group of 93 healthy young dental students. The              occurring in the resident microbial population from
                     results obtained with the Rapid ID 32 A system              oral and nasopharyngeal microbiota in smokers are
                     showed very good to excellent identification for all        correlated with higher frequencies of infections than
                     but 6 strains. The identification at species level of the   in non-smokers [40]. Oropharyngeal flora varies due
                     6 strains mentioned above indicated P. denticola as         to many other factors, such as age and health status.
                     the first choice and Prevotella oralis as the second        In most studies focusing on the upper respiratory
                     choice. These isolates were considered to belong to         tract flora, mainly oral and nasopharyngeal flora
                     P. denticola because P. oralis is known to not produce      have been investigated in children, the elderly and
                     colonies with dark pigment. Atypical phenotypic             patients suffering from various diseases [41-47].
                     characteristics of isolates can sometimes lead to           However, updated data on changes in oropharynx
                     misidentification of species. Therefore, the culture-       flora in healthy young adults are also needed.
                     independent methods are highly recommended for              Influenced by data from recent specialty literature,
                     the detection of anaerobic species. However, both           special attention was paid in the present research
                     culture-based method and detection by molecular             to analyze the association between smoking and
                     techniques also have advantages and limitations.            colonization of oropharynx with black-pigmented
                     Previous studies performed in adults and children           Gram-negative anaerobic bacilli in the group
                     reported P. melaninogenica in a higher percentage           of healthy young adults from whom the strains
                     in patients with tonsilar crypts who suffer from            were isolated. Although the findings showed
                     recurrent tonsillitis, compared to healthy subjects         no statistically significant correlation, for a more
                     with adenotonsillar hyperplasia [17]. Recent studies        accurate interpretation of the results, this study
                     reported that P. melaninogenica colonizes the tonsils       should be continued on a larger sample size.
                     in large quantities [30]. Isolates of this species are      It is important to understand that maintaining
                     usually beta-lactamase producers [31] and this may          a normal oropharyngeal and nasopharyngeal
                     explain the failure of penicillin treatment in recurrent    microbiota is an important step in maintaining good
                     tonsillitis [32]. The bacteria that produce enzymes         health.
                     that destroy the beta-lactam ring of penicillin
                     indirectly protect penicillin-sensitive bacteria such       5. CONCLUSIONS
                     as S. pyogenes [33]. Changes in the oropharyngeal
                     ecology can variably affect the oropharyngeal               The findings of this study indicated P. denticola and
                     microbiota. Smoking is one of the main external             P. melaninogenica as the main species of black-
                     factors that can directly influence the composition         pigmented Gram-negative anaerobic bacilli that
                     of the upper airway microbiota [2]. Both active and         colonize oropharynx in healthy young adults. The
                     passive smoking can contribute to colonization              well-known beta-lactamase activity of these bacteria
                     of the upper respiratory tract with pathogenic              should be strongly considered when antibiotics are
                     microorganisms [34,35] by impairing the immune              needed in oral infection therapy.
                     response and favoring bacterial colonization, either
                     by stimulating microbial adhesion to the epithelium         CONFLICT OF INTEREST
                     or by other means [36,37]. Prevotella comprises             The authors declare no conflict of interest.
                     commensal species from the human microbiota,
                     which usually protect the upper respiratory tract           AUTHOR CONTRIBUTIONS
                     against colonization of pathogenic bacteria [24,35].
                     Some studies have reported that nasopharynx in              All authors had equal contribution to the paper.
                     smokers frequently hosts Streptococcus pneumoniae,
                     Haemophilus influenzae and Moraxella catarrhalis,           ACKNOWLEDGEMENTS
                     while Prevotella is absent [24,35]. It has been
                     observed that the oral cavity and the nasopharynx           This study was part of the internal research plan for 2018 of the
                     became colonized again with the normal resident             Department of Microbiology, Faculty of Dental Medicine, "Carol
                     flora when smoking was abandoned [38,39]. Recent            Davila" University of Medicine and Pharmacy (CDUMPB) and was
                     research using univariate analysis and machine              carried out in co-operation with the Head of the Department of
                     learning approaches has concluded that smokers              Embryology and a member of the Department of Epidemiology,
                     compared to non-smokers are colonized with a                "Carol Davila" University of Medicine and Pharmacy (CDUMPB).




    90               Stoma Edu J. 2020;7(2): 87-93                                                  pISSN 2360-2406; eISSN 2502-0285
Black-pigmented anaerobes that colonize the oropharynx
                                                                                                                                                  www.stomaeduj.com



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                                                                                                           Gabriela BĂNCESCU
                                                                                                    MD, MSc, PhD, Professor, Head
                                                                         Department of Microbiology, Faculty of Dental Medicine
                                                                    “Carol Davila” University of Medicine and Pharmacy Bucharest
                                                                                                               Bucharest, Romania



                     CV
                     Professor Gabriela Băncescu is the Head of the Microbiology Department, Faculty of Dental Medicine, “Carol Davila” University
                     of Medicine and Pharmacy Bucharest, Bucharest, Romania. She was awarded a Master’s degree in Oral Microbiology by the
                     University of Bergen, Norway. She was a member of the board of the European Society of Chemotherapy - Infectious Diseases.
                     She is a member of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and was a member of ESCMID
                     Study Group for Anaerobic Infections. She was the Secretary General of the Romanian Society of Laboratory Medicine (2009-
                     2014) and a member of the editorial board of the Bacteriol Virusol Parazitol Epidemiol, Infectio.ro and the Roum Arch Microbiol
                     Immunol. At present, she is an Editor of the Stoma Edu J and Associate Editor of Rom J Rhinol.




    92               Stoma Edu J. 2020;7(2): 87-93                                                             pISSN 2360-2406; eISSN 2502-0285
Black-pigmented anaerobes that colonize the oropharynx
                                                                                                          www.stomaeduj.com




Questions




                                                                                                        Original Articles
1. Prevotella originated from the genus?
qa. Bacteroides;
qb. Fusobacterium;
qc. Tannerella;
qd. Porphyromonas.

2. Prevotella comprises species of?
qa. Gram-positive anaerobic bacilli;
qb. Gram-negative anaerobic bacilli;
qc. Gram-negative anaerobic cocci;
qd. Gram-negative aerobic bacilli.

3. Exemples of black-pigmented anaerobic species?
qa. Prevotella melaninogenica, Prevotella oralis and Prevotella nigrescens;
qb. Porphyromonas gingivalis, Porphyromonas endodontalis and Prevotella oralis;
qc. Prevotella melaninogenica, Prevotella intermedia and Prevotella nigrescens;
qd. Prevotella intermedia, Porphyromonas gingivalis and Prevotella oralis.

4. The beta-lactamase?
qa. Is never produced by black-pigmented anaerobic strains;
qb. Enhances the penicillin effect against anaerobic bacteria;
qc. Enhances the penicillin effect against aerobic bacteria;
qd. Cleaves the beta-lactam ring of beta-lactam antibiotics.




Stoma Edu J. 2020;7(2): 87-93                                        pISSN 2360-2406; eISSN 2502-0285      93