Nasal colonization and antimicrobial susceptibility pattern of Staphylococcus aureus among pre-school children in Ethiopia
Reta et al. BMC Res Notes
Nasal colonization and antimicrobial susceptibility pattern of Staphylococcus aureus among pre-school children in Ethiopia
Alemayehu Reta 0 1
Moges Wubie 1 2
Getnet Mekuria 1 2
0 Department of Medical Laboratory Science, College of Health Sciences, Debre Markos University , Pobox-269, Debre Markos , Ethiopia
1 Pen penicillin , Amp ampicillin, Aug augmentin, Tet tetracycline, Dox doxycycline, Chl chloramphenicol
2 Department of Public Health, College of Health Sciences, Debre Markos University , Debre Markos , Ethiopia
Background: Staphylococcus aureus is one of the bacterium that can asymptomatically colonize the human upper respiratory tract (i.e. nose and throat). Carriage of S. aureus, including methicillin resistant S. aureus, is common to children. The aim at this study was to determine the nasal colonization, associated factors and antimicrobial susceptibility patterns of S. aureus isolates among pre-school children in Debre Markos town. Methods: Institutional-based cross sectional study was conducted. A total of 400 nasal swabs were collected from pre-school children from April to June, 2015 following standard microbiological methods. MRSA was detected using both Cefoxitin (30 μg) and Oxacillin (6 μg) (Oxoid Ltd. England) discs in combination and associated factors were assessed using self-administered pretested questionnaires, which were delivered to the children's parents/guardians. Statistical analysis of the data (logistic regression) was done using SPSS V-22. Results: A total of 52 S. aureus isolate was recovered from 400 nasal swap samples. The prevalence of S. aureus among pre-School children was 13% (52/400). The susceptibility patterns of the isolates to commonly used antibiotics were: 84.62% to Chloramphenicol, 69.2% to Doxycycline and Tetracycline, 92.3% to Kanamycin, 7.7% to Ampicillin and Penicillin, 86.6% to Ceftriaxone, and 76.9% to Augmentin. All the isolates were sensitive to Oxacillin and Cefoxitin, mean there was no methicillin resistant S. aureus isolate, and also sensitive to Gentamycin, Erythromycin and Clindamycin. The main associated factors of nasal colonization of S. aureus in the study area was, having recurrent acute otitis media (AOM) [AOR = 2.37 (1.11, 5.06)], Children admission in hospital [AOR = 1.96 (1.03, 3.73)] and cough [AOR = 2.09 (1.08, 4.09)]. Conclusions: The prevalence of S. aureus nasal colonization among pre-school children was relatively low in absence of MRSA. Factors like; recurrent AOM, hospital admission and cough were significantly associated with S. aureus nasal colonization. Most of the isolates were resistant to β-lactam drugs and sensitive to drugs like Gentamycin, Erythromycin, Clindamycin, Chloramphenicol, Doxycycline, Tetracycline, Kanamycin and Augmentin.
Antimicrobial susceptibility pattern; Staphylococcus aureus; Nasal colonization; Pre-school children
Nasal colonization of S. aureus creates a risk factor of
developing invasive disease. Children are commonly
considered to be persistent carriers more frequently
than adults, with the highest rate of carriage reached
in the first year of life and transition from persistent to
intermittent or non-carriage occurring during
There is increasing evidence that S. aureus nasal
colonization is spreading among healthy pre-school children.
Studies conducted around the world reveal that, 17.5% in
], 34% in Belgium [
], 35% in India [
Republic of Korea [
], 48% in Brazil [
], and 17.3% in Turkey
] of pre-school children were colonized by S. aureus,
even if the associated factors are not readily identified.
While some studies have demonstrated recurrent acute
otitis media (AOM), frequent exposure to antibiotics [
socioeconomic status [
], healthcare worker in the home
], child-care attendance [
], hospitalization, chronic
illness and previous isolation of methicillin resistant S.
aureus (MRSA) [
] but there is no definite consensus as
to why healthy children acquire these resistant bacteria
]. There are few studies conducted to assess nasal
colonization of S. aureus among pre-school children around
the globe and to our best knowledge, there is no study
conducted on factors associated with nasal
colonization of S. aureus particularly among pre-school children
in the study area and in Ethiopia at large. Therefore this
study will create a better understanding of the
epidemiology and determinants of S. aureus nasal colonization
could positively be helpful in improving infection control
and prevention strategies and designing protocols.
Study enrollment, specimen collection and processing
Institution based cross sectional study was conducted at
kindergarten school from April to June, 2015. All
sampled pre-school children (1–6 years) who were attending
their kindergarten education in Debre Markos town
during the study period were selected by using simple
random sampling technique. Children were excluded from
the study, if they had received antibiotic treatment for the
previous 7 days. Individual variables were obtained from
the children’s guardians/parents immediately after
written consent is obtained from the guardians/parents and
before swabs were collected. After written informed
consent was obtained, each child had a specimen collected
from the anterior nares with a dry, sterile and moistened
swab (Ventura Tran system; Copan Diagnostics, Corona,
Calif.). The tip of the collection swab was inserted 2–3 cm
into the anterior nares and rotated 4–5 times both
clockwise and anticlockwise before withdrawal in each nostril.
Swabs were placed into Screw capped test tubes
containing enrichment broth (contained 37.5 g sodium chloride
(NaCl), 1.25 g yeast extract, 5.0 g tryptone, and 500 ml
distilled water) and the samples were labeled, packaged
and transported to the Medical Microbiology Laboratory
of Debre Markos University within 2 h of collection and
processed immediately according to standard
Microbiological procedures [
Selective media and culture conditions
Each 10 μl of incubated enrichment broth was inoculated
in both blood agar and MSA (Oxoid Ltd., England) and
incubated at 35 °C for 24–48 h. Golden yellow colonies
of the MSA and white creamy colonies in blood agar
indicate S. aureus, which was subsequently identified by
gram staining, 3% catalase testing, tube coagulase testing
with the Staph Latex agglutination assay (Life Sign,
Somerset, N.J.) [
Identification of MRSA
Staphylococcus aureus isolates were tested for
Methicillin resistance by using modified Kirby-Bauer disc
diffusion technique [
]. Inocula were adjusted to 0.5%
McFarland standard and each streaked uniformly with
the swab sticks in Mueller–Hinton agar plates
containing 6.5% NaCl to obtain confluent growth [
]. The plates
were allowed to dry for 3–5 min before the disks used.
The Oxacillin (6 µg) and Cefoxitin (30 µg) discs were
used for susceptibility testing. The plates then incubated
aerobically at 35 °C for 24 h. The results were interpreted
according to Clinical and Laboratory Standard Institute
(CLSI) guidelines for Oxacillin and Cefoxitin
susceptibility testing: a zone size of < 10 mm was considered
resistant; a zone size of > 13 mm was considered susceptible
for Oxacillin and a zone size of < 21 mm was considered
resistant; a zone size of > 22 mm was considered
susceptible for Cefoxitin [
Antibiotic susceptibility testing
The antibiotic susceptibility testing was performed by
modified Kirby-Bauer disc diffusion technique and
MIC for Ceftriaxone [
]. The colonies were picked up
with wooden applicator stick and dipped into nutrient
broth to make direct colony suspension of the isolates
and inocula were adjusted at 0.5% McFarland standard.
After few minutes these suspensions were streaked with
Mueller–Hinton agar plates. The antibiotic susceptibility
testing was performed on the following antibiotic discs;
Ceftriaxone (30 μg), Chloramphenicol (30 μg),
Clindamycin (2 μg), Erythromycin (15 μg), Gentamicin (10 μg),
Penicillin G (10 units), Doxycycline (30 μg), Tetracycline
(30 μg), Kanamycin (30 μg), Amoxicillin–Clavulanic acid
(20 μg), Cefoxitin (30 µg), Ampicillin (10 μg) and
Oxacillin (6 μg). All antibiotic discs/drugs were obtained from
Oxoid Ltd. England. The plates were incubated at 37 °C
for 18–24 h.
The disc diffusion technique and zone interpretation of
each antimicrobial agent were used in accordance with
CLSI guidelines [
]. Diameters of the zone of inhibition
around the disc were measured using a graduated caliper
in millimeters and the isolates was classified as sensitive,
intermediate, and resistant according to the
standardized table supplied by the CLSI. Staphylococcus aureus
(ATCC-25923) was used as control for the antimicrobial
The collected data were checked for completeness, coded,
and then entered into Epi data version 3.1 software for an
edition and cleaning of data and export to SPSS version
22 Software for analysis. Bivariate and multivariate
logistic regression analysis were performed to identify factors
associated with nasal colonization of S. aureus.
Of the 422 eligible parents/guardians, 400 agreed to
participate in the study, which made a response rate
of 94.8%. The median age of pre-school children were
5 years (Inter quartile range, IQR ± 3). With regard to
household monthly income, the median income were
3100 birr (Inter quartile range, IQR ± 16, 267). More
than half (55%) of households had income of 2001–5000
birr monthly (Table 1).
Prevalence of S. aureus
About 74% (296/400) of the pre-school children involved
in the study were aged > 4 years, of these 12% (48/400)
were colonized by S. aureus. Majority of the study
participants were females, which accounts 60% (240/400)
among them 8% (32/400) were colonized by S. aureus.
The overall prevalence of S. aureus were 13% (52/400)
(Table 2). The good news from our study was, there was
no MRSA isolates among S. aureus colonized pre-school
Medical and related characteristics
The majority (87.8%) of pre- school children didn’t
colonize by S. aureus. Fifty-seven (14.3%) of pre-school
children had recurrent AOM. More than half (52.8%) of
pre-school children had Cough in the previous 30 days.
One-fourth (25.3%) of pre-school children was admitted
to hospital in the previous 12 months (Table 3).
Factors associated with nasal colonization of S. aureus
Hosmer–Lemeshow goodness-of-fit tested (p = 0.425)
was used to assess the fitness of the model. During the
bivariate logistic regression analysis, child admission
to hospital in the previous 12 months, Skin/soft tissue
infection, having recurrent AOM, cough in the
previous 30 days, family member with wound in the previous
12 months and child use of antibiotic in the previous
12 months were significantly associated with nasal
colonization of S. aureus.
During the multivariate logistic regression analysis,
child admission to hospital in the previous 12 months,
having recurrent AOM and cough in the previous 30 days
was significantly associated with nasal colonization of S.
Children who were admitted in hospital in the
previous 12 months were 1.96 times more likely colonized by
S. aureus than those children not admitted [AOR = 1.96
(1.03,3.73)]. Those children that had recurrent AOM
were 2.37 times more likely colonized by S. aureus than
children hadn’t recurrent AOM [AOR = 2.37 (1.11,5.06)].
Those children that had coughed in the previous 30 days
were 2.09 times more likely colonized by S. aureus
than children hadn’t coughed [AOR = 2.09 (1.08,4.09)]
Evaluation of antimicrobial susceptibility tests
The in vitro antimicrobial susceptibility pattern
results in 52 S. aureus isolates were shown in Table 2.
A total of 52 S. aureus isolates were subjected to
Presence of S. aureus
antibiotic susceptibility test against 13 antimicrobial
drugs (Table 5). The susceptibility patterns of the isolates
to commonly used antibiotics were: 84.62% to
Chloramphenicol, 69.2% to Doxycycline and Tetracycline, 92.3%
to Kanamycin, 7.7% to Ampicillin and Penicillin, 86.6%
to Ceftriaxone (but after performing minimum
inhibitory concentration (MIC), all the isolates become
susceptible for Ceftriaxone), and 76.9% to Augmentin. All the
isolates were sensitive to Oxacillin and Cefoxitin, mean
there was no MRSA isolate and also sensitive to
Gentamycin, Erythromycin and Clindamycin.
Drug resistance pattern/antibiogram of S. aureus
The resistance patterns of S. aureus isolates varied from
one to six antimicrobial drugs. The highest resistance
pattern (46.15%) was observed for two antibiotics with
pattern of Penicillin/Ampicillin. Three resistance
patterns of equal proportion were observed for resistance
to one, three and six antibiotics such as
Chloramphenicol, Penicillin/Ampicillin/Tetracycline and Penicillin/
Ampicillin/Augmentin/Tetracycline/Doxycycline/Chloramphenicol, respectively and also Penicillin/Ampicillin/
Penicillin/Ampicillin/Tetracycline/Doxycycline had the same resistance pattern proportion.
Maximum resistance (resistant to six drugs) was observed for
one S. aureus isolate from resistance pattern of Penicillin/
Ampicillin/Augmentin/Tetracycline/Doxycycline/Chloramphenicol. Multi-drug resistance (defined as resistance
to at least three different antibiotic groups) S. aureus
isolates were observed (Table 6).
In the present study, the overall frequency of isolation
of S. aureus from pre-school children of Debre Markos
town was 13% (52/400). In contrast with this, a study
conducted in Belgium, [
], around 50% of pre-school
children were colonized with S. aureus. This study also
performed a prospective cohort study, how
socio-economic status affects S. aureus nasal colonization among
preschool children, and conclude that there is high S.
aureus nasal colonization among preschool children
that have high socio-economic status when we
compare with low socio-economic status. As we know our
country, Ethiopia is one of a developing country having
low socio-economic status. This factor may be the reason
why the current study obtained decreased S. aureus nasal
colonization among pre-school children in Debre Markos
town. In addition to this, seasonal variations,
geographical distribution and method of isolation may affect the
In line with the current study, a study conducted in
], the prevalence of S. aureus nasal
colonization among children attending day care center having
1 month–5 year ages was 17.9% and also a study
conducted in Turkey, [
] reveals that, 17.3% of pre-school
children were colonized by S. aureus.
The susceptibility patterns of the isolates to commonly
used antibiotics were: 84.62% to Chloramphenicol, 69.2%
to Doxycycline and Tetracycline, 92.3% to Kanamycin,
7.7% to Ampicillin and Penicillin, 86.6% to Ceftriaxone,
and 76.9% to Augmentin. All the isolates were sensitive
to Oxacillin, Gentamycin, Erythromycin and
Clindamycin. The findings of our current study were similar with a
study conducted in Turkey [
], all S. aureus isolates were
sensitive to Gentamycin, Erythromycin and Clindamycin.
Our finding showed that, there is no methicillin
resistant S. aureus isolates. This finding is in agreement with
a study conducted in Turkey, [
], they isolated only one
MRSA (0.1%) from a newborn infant that had been
hospitalized during the first week of his life in the neonatal
intensive care unit, which may suggest hospital acquired/
The prevalence of carriage and the effect of various
epidemiological factors of nasal colonization of S. aureus
has been described, but results vary from one study to
another, in parallel with differences in the populations
included or definitions used.
A study conducted in India shows that, attending
kindergarten schools or being in pre-school are one factor
to be colonized with S. aureus [
]. Through such
investigation these pre-school children could be target groups
to undergo further assessment on factors related to nasal
colonization of pre-school children with S. aureus. In
our current study, the main associated factors of nasal
colonization of S. aureus among pre-school children was
Child admitted to hospital in the previous 12 months
[AOR = 1.96 (1.03, 3.73); p = 0.040], having recurrent
AOM [AOR = 2.37 (1.11, 5.06); p = 0.026] and Cough in
the previous 30 days [AOR = 2.09 (1.08, 4.09); p = 0.029].
A study conducted among North Carolina children
states that, antibiotic use by children in the past 6 months
were associated with nasal colonization of S. aureus.
In addition to that, the study also reveals that, parental
employment in a school/daycare and family history of
boils were associated with S. aureus nasal carriage [
In line with that, our study also obtained family member
with wound in the previous 12 months were associated
with nasal colonization of S. aureus in the bivariate
logistic regression analysis.
Concerning about recurrent AOM in the previous
12 months, a study conducted in Ethiopia by the
corresponding author of this research (Alemayehu Reta)
shows statistically significant association among school
In agreement with our finding, a study conducted in
Nepal by Rijal KR and his colleagues illustrates that,
children admitted to hospital in the past 12 months were
statistically associated with nasal colonization of S. aureus
] and also a study in Turkey children shows a similar
finding, which is conducted among children having the
age of 6 months up to 15 years, hospitalization or
surgical operation in the previous 1 year were associated with
S. aureus colonization [
] obviously, there are infectious
agents in the hospital environment that can be
considered as hospital acquired infection. S. aureus is one of the
nosocomial infectious agent that can disseminate to the
community when an admitted patient is discharged. So
if the child is admitted previously, there will be a greater
probability of being colonized by S. aureus.
Conclusions and recommendations
This is the first study among Ethiopian pre-school
children below 6 years of age studying nasal colonization of S.
aureus and antimicrobial susceptibility pattern of the
isolates and showed a relatively low prevalence of S. aureus.
Most of the isolates were resistant to β-lactam drugs and
sensitive to drugs like Gentamycin, Erythromycin,
Clindamycin, Chloramphenicol, Doxycycline, Tetracycline,
Kanamycin and Augmentin. The study shows that
attending pre-school was associated with nasal colonization
of S. aureus. Studies with cohort design plus molecular
analysis of the isolates are needed to accurately assess the
epidemiology of S. aureus nasal colonization in various
geographical locations, to see the strain distribution and
to detect heteroresistant isolates. More studies on
additional associated factors with a large sample size, which
accompanies various geographical locations should be
done for a better understanding of S. aureus nasal
colonization and to design preventive strategies that target
bacterial colonization in the upper respiratory tract (throat,
AOM: acute otitis media; AOR: adjusted odds ratio; ATCC: American Type
Culture Collection; CI: confidence interval; CLSI: Clinical and Laboratory Standard
Institute; IQR: inter quartile range; MIC: minimum inhibitory concentration;
MRSA: methicillin resistant Staphylococcus aureus; MSA: mannitol salt agar;
SPSS: Statistical Package for the Social Science.
AR carried out bacteriological studies, participated in the Microbiological
analysis of samples, interpretation of data, drafted the manuscript, revising it
critically for important intellectual content, given final approval of the version
to be published. GM participated in acquisition of funding, collection of data
and statistical analysis. MU participated in acquisition of funding and general
supervision of the research group. All authors read and approved the final
AR Medical Microbiologist, department head of Medical Laboratory Science,
reviewer, researcher, give community service, advisor ship, and academic
staff. GM Human Nutritionist, MU Infectious Disease, all perform activities like:
research, give community service, advisor ship, and academic staff.
We would like to acknowledge Debre Markos University for funding, Molla
Yigzaw for sample collection and Getiye Dejenu for his contribution during
The authors declare that they have no competing interests.
Availability of data and materials
The datasets generated and/or analyzed during the current study are not
publicly available due a restriction by the University research legislation and
some ethical issue but are available from the corresponding author on
Consent to publish
Ethics approval and consent to participate
Ethical clearance was obtained from Debre Markos University ethical review
board. Letter of permission to conduct the study was obtained from the
director of each school. Informed consent was obtained from each study
participant’s guardian/parent. The purpose of the study was clearly described
to the study participants including the benefits and risks of the study. Any
information about the participants was kept confidential and the specimen
collected from the participants was only analyzed for the intended purposes.
The participants were enrolled only after they are sufficiently counseled and
their informed consents have been obtained. Relevant confidentiality was
maintained for every participant. After analysis of the participant’s specimen,
the positive findings for S. aureus were decolonized by using 2% Muprocin.
The research was funded by Debre Markos University and we declare that the
funder do not participate in the design of the study and collection, analysis,
and interpretation of data and in writing the manuscript.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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