IDENTIFICATION AND ANTIBIOGRAM OF BACTERIA FROM USED TOOTHBRUSHES BY STUDENTS OF FEDERAL UNIVERSITY WUKARI, NORTH EAST, NIGERIA

Abstract

Toothbrushes play a significant role in disease transmission and increase the risk of infection since they can serve as a reservoir for microorganisms. This study was carried out to investigate the presence of bacteria on regularly used toothbrush. One hundred and twenty (120) in-use toothbrushes were used for this study. Sixty (60) pieces each from males and female; with half the number obtained from both the males and females living within the school hostel and males and females living off school campus were examined using standard bacteriological techniques. The results showed a total of six (6) bacteria isolates; Staphylococcus aureus 52%, Streptococcus species 22%, Staphylococcus epidemidis 11%, Pseudomonas aeruginosa 9%, Escherichiacoli 4%, and Klebsiella oxytoca 2% which clearly showed Staphylococcus aureus as the most commonly isolated bacteria from toothbrushes in the localities. The antibiogram of the isolates show that all isolates were susceptible to ciprofloxacin and resistant to augmentin. Other antibiotics (gentamycin, streptomycin, chloramphenicol and ampiclox) has various degree of susceptibility and resistant. Also, the presence of these bacteria in the various toothbrushes samples investigated could have been as a result of exposure to dirty environment, contaminated water or all left over materials from food consumed. In conclusion, the presence and multiplication of the above bacterial in toothbrushes may lead to infection and decaying of teeth and hence lead to smelling mouth and breath. Therefore, whenever there is decaying of teeth or mouth infection, especially one which delays in healing, routine culture should be carried out to determine bacterial associated with such decay and its susceptibility to various antibiotics should also be carried out to determine the choice of antibiotic for treatment. Hence good hygiene and proper care of toothbrushes plus in-cooperation of antimicrobial drugs during mouth wash is advised.

Full text

FUW Trends in Science & Technology Journal, www.ftstjournal.com
e-ISSN: 24085162; p-ISSN: 20485170; December, 2019: Vol. 4 No. 3 pp. 657 – 659
657
IDENTIFICATION AND ANTIBIOGRAM OF BACTERIA FROM USED
TOOTHBRUSHES BY STUDENTS OF FEDERAL UNIVERSITY WUKARI,
NORTH EAST, NIGERIA
E. P. K. Imarenezor*, S. T. C. Brown, F. U. Ebuara and S. Sani
Tropical Diseases Unit, Department of Microbiology, Federal University Wukari, Nigeria
*Corresponding author: kimarenezor@yahoo.com
Received: June 18, 2019 Accepted: September 06, 2019
Abstract: Toothbrushes play a significant role in disease transmission and increase the risk of infection since they can serve
as a reservoir for microorganisms. This study was carried out to investigate the presence of bacteria on regularly
used toothbrush. One hundred and twenty (120) in-use toothbrushes were used for this study. Sixty (60) pieces
each from males and female; with half the number obtained from both the males and females living within the
school hostel and males and females living off school campus were examined using standard bacteriological
techniques. The results showed a total of six (6) bacteria isolates; Staphylococcus aureus 52%, Streptococcus
species 22%, Staphylococcus epidemidis 11%, Pseudomonas aeruginosa 9%, Escherichiacoli 4%, and Klebsiella
oxytoca 2% which clearly showed Staphylococcus aureus as the most commonly isolated bacteria from
toothbrushes in the localities. The antibiogram of the isolates show that all isolates were susceptible to
ciprofloxacin and resistant to augmentin. Other antibiotics (gentamycin, streptomycin, chloramphenicol and
ampiclox) has various degree of susceptibility and resistant. Also, the presence of these bacteria in the various
toothbrushes samples investigated could have been as a result of exposure to dirty environment, contaminated
water or all left over materials from food consumed. In conclusion, the presence and multiplication of the above
bacterial in toothbrushes may lead to infection and decaying of teeth and hence lead to smelling mouth and breath.
Therefore, whenever there is decaying of teeth or mouth infection, especially one which delays in healing, routine
culture should be carried out to determine bacterial associated with such decay and its susceptibility to various
antibiotics should also be carried out to determine the choice of antibiotic for treatment. Hence good hygiene and
proper care of toothbrushes plus in-cooperation of antimicrobial drugs during mouth wash is advised.
Keywords: Bacteria, identification, students toothbrushes, University, Wukari
Introduction
A toothbrush is a dental instrument used for cleaning teeth,
ideally in conjunction with toothpaste or mouthwash. The
toothbrush consists of a plastic handle and nylon bristles
attached to the head of the brush. A toothbrush plays a
pertinent role in oral hygiene and it is commonly found in
homes and other places of human residence. It could also play
a significant role in disease transmission since it can serve as
reservoir for microorganisms in healthy and medically ill
adults. Many bacteria are found on toothbrushes after
brushing. These microorganisms can remain viable for a day
to a week after brushing (Efstratius et al., 2007; Downes et
al., 2008). Toothbrushes are most commonly located near the
bathroom sink, which is very conducive for microbial growth.
A new toothbrush is usually not a favourable habitat for
bacteria, but in some cases, it is already slightly contaminated
because of the absence of regulations that ensure its sterility
when packaged for sale (Glass, 2012). The mouth is a
hospitable niche for all kinds of microbes and thus, the
toothbrush will always be contaminated through brushing
(Quirynen et al., 2011).
Oral diseases can be greatly controlled by reducing the
microbial load in the oral cavity and this can be achieved by
maintaining proper oral hygiene (Karibasappa et al., 2011).
The human oral cavity is colonized by a larger variety of
bacteria flora than any other anatomic area. More than seven
hundred (700) species of bacteria have already been
identified, four hundred (400) of which were found in the
periodontal pocket adjacent to teeth. Organisms not normally
associated with oral flora also have been isolated from
toothbrushes including enterobacteria, Pseudomonads
(Sammons et al., 2004). The infectious microorganisms
remaining on the brushcan re-infect our mouth and teeth
again, with some of them even spreading to the rest of our
body and causing serious health problems, including heart
disease, stroke, arthritis etc. (Warren et al., 2001).
A new toothbrush is usually not a favourable habitat for
bacteria and fungi, but in some cases, toothbrushes are already
slightly infected because no regulation that stating that
toothbrushes must be sold in a sterile packages exist
(Efstratius et al 2007). Tooth brushing plays an important
everyday role for personal oral hygiene and effective plaque
removal. Appropriate toothbrush care and maintenance are
also important considerations for sound oral hygiene. The oral
cavity is home to hundreds of different types of
microorganisms (Mehta et al., 2007). Therefore, it is not
surprising that some of these microorganisms are transferred
to a toothbrush during use. It may also be possible for
microorganisms that are present in the environment where the
toothbrush is stored to establish them on the brush, since they
are not required to be sold in a sterile package (Dabas et al.,
2008). The toothbrush is not naturally favourable towards the
growth of microbes, but can sustain bacterial life once they
are transferred onto the toothbrush. Different modes of
transfer are responsible for the bacteria on the toothbrush such
as contact with the mouth, cross contamination, and the
bacteria in the toilet community (Alm et al., 2007). The
organisms that can survive for a certain amount of time on the
toothbrush are diverse, ranging from fungus to bacteria to
yeast. The environment of the toothbrush is affected by many
conditions whether it is the architecture of the toothbrush
itself regarding bristles or by adjusting the pH level. These
conditions alter the population of bacteria on the toothbrush.
While the toothbrush is not the ideal niche for a microbe, the
toothbrush is capable of supporting microbial life (Ismail et
al., 2007). Toothbrushes are necessary for daily oral hygiene,
but residues remaining on their bristles may precipitate the
growth of several microorganisms. Oral biofilms develop over
time into exceedingly complex communities.
Hundreds of species of bacteria has been identified in such
biofilms (Johansson et al., 2009). The oral cavity, the skin,
and the upper respiratory tract are the primary portals for
Streptococcus viridans, Staphylococcus speciesand
Supported by

Investigation of the Presence of Bacteria on Regularly Used Toothbrush
FUW Trends in Science & Technology Journal, www.ftstjournal.com
e-ISSN: 24085162; p-ISSN: 20485170; December, 2019: Vol. 4 No. 3 pp. 657 – 659
658
Haemophilus aphrophilus, Aggregatibacter (formerly
Actinobacillus) Actinomycetem comitans, Cardiobacterium
hominis, Eikenella corrodens, and Kingella kingae (HACEK)
organisms with streptococcal and staphylococcal organisms
responsible for more than 80% of cases of bacterial
endocarditis (Imarenezor et al., 2016). The overall survival
rate for patients with native valve endocarditis caused by
Streptococcus viridans, HACEK organisms, or enterococci
ranges from 85-95%. For Staphylococcus aureus native valve
endocarditis, the mortality rate is 55-70% in persons who do
not abuse intravenous drugs and is 85-90% in those who do.
Prosthetic valve endocarditis beginning within 2 months of
valve replacement results in mortality rates of 40-50% (Burt et
al., 2006). The administration of antibiotic prophylaxis to at-
risk patients who are undergoing dental manipulations is a
reasonably well accepted clinical practice (Cook et al., 2008).
Numerous studies have demonstrated that antibiotics can
reduce the prevalence and the magnitude of bacteremia
(Baltch et al., 2012).
Today, mutansstreptococci are considered to be the main
aetiological microorganisms in caries disease, withlactobacilli
and other microorganisms participating in the disease
progression. The mouth is home to millions of
microorganisms (germs). In removing plaque and other soft
debris from the teeth, toothbrushes get contaminated with
bacteria, blood, saliva, oral debris, and toothpaste. Because of
this contamination, a common recommendation is to rinse
one's toothbrush thoroughly with tap water following
brushing. Limited research has suggested that even after being
rinsed visibly clean, toothbrushes can remain contaminated
with potentially pathogenic organisms. In response to this,
various means of cleaning, disinfecting or sterilizing
toothbrushes between uses have been developed. This
research is therefore aim at isolation and identifying the
various contaminating bacterial isolates on regularly used
toothbrush.
Materials and Methods
Study area
This study was carried out in the Department of
Microbiology, Federal University Wukari, Taraba State,
Nigeria. Wukari metropolis is a large town which is the
Headquarter of Wukari Local Government Area of Taraba
State. Geographically, Wukari lies between latitude 7°55’42”
North and longitude 9°47’59” East. It has an area of 4,308
km2 and a population of about thirty thousand (30000).
Wukari is home to Federal University Wukari and Kwararafa
University. The major languages spoken are Jukun, Kutep,
Tiv, Hausa and Fulani. The population is dependent on factors
such as migration and economy. The inhabitants of Wukari
are mostly farmers while a few indulge in commerce and civil
service (Imarenezoe et al., 2016).
Collection of samples
One hundred and twenty (120) samples of already in–use
toothbrushes were randomly collected aseptically from the
students. The students only agreed to release their
toothbrushes upon receiving a new replacement. The samples
were labeled accordingly and taken to the laboratory for
investigation
Sample preparation
The head region of the toothbrushes was cut off with a sterile
scissor according to Sammons et al. (2004) standard and
soaked in a 10 ml peptone water solution for 60 min. After
which they were vortexed slowly for a minute to dislodge
adherent bacteria from the samples.
Culture procedure
The bacterial suspension was one fold diluted for 10 and (0.1
ml) of broth was plated out by use of a sterile pipette into
MacConkey agar (MCA), Nutrient agar (NA), and Trypton
soy agar (TSA) where NA served as the non-selective
medium, MCA served as a selective media for the isolation of
enterobacteria and TSA for staphylococci and other Gram-
positive bacteria.
Results and Discussion
The occurrence and percentage of the bacterial isolates from
used toothbrushes is shown on Table 1 while Table 2
indicated numbers of contaminated toothbrushes according to
sex and the bacteria isolated. Tables 3 and 4 give the overall
cultural and biochemical characteristics of bacteria isolates
from the various toothbrushes and antimicrobial susceptibility
test of bacterial isolates against selected antibiotics,
respectively.
Table 1: Occurrence and percentage of the bacterial
isolates from used toothbrushes
Bacterial isolates
Number of
Isolates
% of
Isolates
Staphylococcus aureus
63
(52.5%)
Streptococcus species
26
(21.6%)
Staphylococcus epidermidis
13
(10.83%)
Pseudomonas aeroginosa
11
(9.17%)
Escherichia coli
5
(4.17%)
Klebsiella oxytoca
2
(1.67%)
Table 2: Number of contaminated toothbrushes according to sex
Bacterial isolates
Males
Females
Total
Staphylococcus aureus
31(50)
32(50)
32
Streptococcus species
11(45.5)
15(55.5)
11
Staphylococcus epidermidis
9(62.5)
4(37.5)
8
Pseudomonas aeroginosa
6(50)
5(50)
4
Escherichia coli
1(25)
4(75)
4
Klebsiellia oxytoca
2(100)
0(0)
1
Total specimen
30
30
60
Most of the investigated toothbrushes were heavily dirty with
chopped bristles irreversibly bending away from their normal
positions and with the smell of toothpaste. Of the one hundred
and twenty (120) samples investigated for bacterial
contamination, the result showed that bacteria were isolated
from all the used toothbrushes and these bacterial isolates
were identified as Streptococcus species, Pseudomonas
aeruginosa, Escherichia coli, Klebsiella oxytoca,
Staphylococcus aureus and Staphylococcus epidermidis, this
aligned with the work of Imarenezor et al. (2016). The
susceptibility of the isolated bacteria to a selection of six (6)
antibiotics is shown in varying degree of susceptibility which
showed that ciprofloxacin was the most effective as it
inhibited all the organisms with a 0% resistivity followed by
gentamycin, chloramphenicol and ampiclox with 50%
sensitivity each.

Investigation of the Presence of Bacteria on Regularly Used Toothbrush
FUW Trends in Science & Technology Journal, www.ftstjournal.com
e-ISSN: 24085162; p-ISSN: 20485170; December, 2019: Vol. 4 No. 3 pp. 657 – 659
659
Table 3: Overall cultural and biochemical characteristics of bacteria isolates from the various toothbrushes
S/N
Morphology
Gram stain
coa
cat
Citr
Oxi
Suc
Glu
lac
Gas
H2S
Indole
Organism
1
Pink, round, flat, dry
-ve rod
_
+
_
_
+
+
+
+
_
+
E. coli
2
yellow, round, moist
+ve cocci
+
+
+
_
+
_
+
_
_
_
S. aureus
3
Greenish, round, flat, dry
-ve rod
_
+
+
+
+
_
+
_
_
+
P. aeruginosa
4
whitish, round, moist
+ve cocci
_
+
_
+
+
+
+
+
_
S. epidemidis
5
Creamy, round, flat
-ve rod
_
+
+
_
+
+
+
+
_
+
K. oxytoca
6
Yellow, round, moist
+ve cocci
_
_
_
_
+
+
+
_
_
_
S. species
cit = citrate, oxi = oxidase, coa = coagulase, cat = catalase, glu = glucose, lac = lactose, suc = sucrose, and H2S = hydrogen sulphide, + = positive
and - = negative
The least effective was septrin with a mere 20% sensitivity.
Interestingly, chloramphenicol inhibited all the enterobacteria
(Klebsiella species, Escherichia coli and Pseudomonas
aeruginosa) isolated. Six strains of bacteria were isolated
from the one hundred and twenty (120) toothbrushes
investigated (Table 2). This is probably because the
toothbrushes were poorly stored after use in closed containers
or kept in moist toilet places devoid of solar radiation
(disinfection) and ventilation. This is in agreement with
Sammone et al. (2004) and Baltch et al. (2012). The leading
cause of presence of these bacterial types on toothbrushes
could be due to the moist environment of bathrooms and
toilets especially when these environments are stabilized and
the brush is not aired (Caudry et al., 2015). The occurrence of
the bacterial isolates, on the used toothbrushes is presented in
Table 2. Staphylococcus aureus was most frequently isolated
(52%) followed by Streptococcus species (22%),
Staphylococcus epidermidis (11%), Pseudomonas aeruginosa
(9%), Escherichia coli (4%) while Klebsiella oxytoca had the
least occurrence 2%. These result in agreement with previous
researched work by Warren et al. (2001); Dabas et al. (2008)
and Baltch et al. (2012). The overwhelming availability of
Staphylococcus aureus could be linked to poor storage and
handling by the individuals, this is in agreement also with
Imarenezor et al. (2016).
Table 4: Antimicrobial susceptibility test of bacterial
isolates against selected antibiotics
S/N
Isolate
CIP
AU
CN
SXT
CH
APX
1
E. coli
S
R
S
S
R
R
2
S. aureus
S
R
R
S
S
S
3
P. aeruginosa
S
R
R
R
R
R
4
S. epidermidis
S
R
S
S
S
S
5
K. species
S
R
S
S
R
R
6
S. species
S
R
R
S
S
S
S = Sensitive, R = Resistant, CIP = ciprofloxacin, AU = augmentin,
CN = gentamycin, SXT = septrin, CH = chloramphenicol and
APX=ampicilin/cloxacillin
Conclusion
In conclusion, all the used toothbrushes examined in this
study were contaminated with bacteria, which are known to
cause serious health problems in humans. Since toothbrushes
serve as a reservoir for microorganisms and play a major role
in disease transmission and can also increase the risk of
infections to users, their care should be given adequate
attention. They must be adequately rinsed with good water
and allowed to dry in air before storing in hygienic dry
containers. In addition, disinfection of toothbrushes before use
should be encouraged and sharing of toothbrushes should be
discouraged.
Conflict of Interest
Authors have declared that there is no conflict of interest in
this study.
References
Alm A, Wendt L, Koch G & Birkhed D 2007. Prevalence of a
proximalcariesin posterior teeth in 15-year-old Swedish
teenagers in relation to their caries experience at 3 years
of age. Caries Research, 41: 392-340
Baltch L, Pressman C, Schaffer R, Smith M, Hammer M,
Shayegani & Michelsen P 2012. Bacteremia in patients
undergoing prophylaxis as recommended by the
American Heart Association, 148: 1084–1088.
Burt B, Kolker L, Sandretto A, Yuan Y, Ismail AI & Sohn W
2006. Dietary patterns related to caries in a low-income
adult population. Caries Research, 40(6): 473-480.
Caudry D, Klitorinos A & Chan E 2015 .Contaminated
toothbrushes and their disinfection. Journal of Canadian
Dental Association 61: 511-556.
Cook S, Martinez-Mier E & Dean A 2008. Dental caries
experience and association to risk indicators of remote
rural populations. Int. J. Paediatric Dentistry, 4: 275-
283.
Dabas N 2008. A transcription factor regulatory cascade
controls secreted aspartic protease expression in Candida
albicans. Molecular Microbiology, 3: 586-602.
Downes J, Hooper SJ, Wilson MJ & Wade W 2008.
Prevotellahisticola species. isolated from the human oral
cavity. Int. J. Sys. and Evolutionary Microbio., 8: 1788-
1791.
Efstratiou M, Papaioannou W, Nakou M, Ktenas E, Vrotsos I
& Panis V 2007. Contamination of a toothbrush with
antibacterial properties by oral microorganisms. Journal
of Dentistry, 35: 331-37.
Glass R 2012. The infected toothbrush, the infected denture
and transmission of disease. Comprehensive Continued
Dentistry Education, 8: 592-598.
Imarenezor EPK, Brown STC, Yakubu OE & Soken DC
2016. Survey of hepatitis B and C among students of
Federal University Wukari, Taraba State, Nigeria. Int.
Res. J Medicine and Med. Sci., 4(3): 31-37.
Ismail AI, Sohn W, Tellez M, Awaya A, Sen A, Hasson H
and Pitts N (2007).The International Caries Detection
and Assessment System: an integrated system for
measuring dental caries, 35(3): 170-178.
Johansson I, Larsson B, Nordlund A & Thorild A 2009 Diet
and dental caries. The J. Am. Dental Assoc., 140: 25S-
34S
Karibasappa G, Nagesh L & Sujatha B 2011. Assessment of
microbial contamination of toothbrush head: An in vitro
study. Indian J. Dental Res., 22(1): 2-5.
Mehta A, Sequeira PS & Bhat G 2007. Bacterial
contamination and decontamination of toothbrushes after
use. The New York State Dental Journal, 73: 20–22.
Quirynen M, De Soete M, Pauwels M, Gizani S, Van Meerbeek B
& van Steenberghe D 2011. Can toothpaste or a toothbursh
with antibacterial tufts prevent toothbrush contamination.
Journal of Periodontology, 74: 312–322.
Sammons RL, Kaur D & Neal P 2004. Bacterial survival and
biofilm formation on conventional and antibacterial
toothbrushes. Biofilms, 1: 123-130.
Warren D, Goldschmidt M, Thompson B, Adler-Storthz K &
Kenneth H 2001. The effects of toothpastes on the
residual microbial contamination of toothbrushes.
Journal of American Dentology, 132: 1242-1245.

Investigation of the Presence of Bacteria on Regularly Used Toothbrush
FUW Trends in Science & Technology Journal, www.ftstjournal.com
e-ISSN: 24085162; p-ISSN: 20485170; December, 2019: Vol. 4 No. 3 pp. 657 – 659
656