Original
research
Identification of bacterial isolates from
nasolacrimal duct infection in children with congenital nasolacrimal duct
obstruction from Feiz teaching hospital, Isfahan
Helma Ebneali1,
Fereshteh Saffari2, Alireza Zandi3, Jamshid Faghri1,*
1Department of Microbiology, School of Medicine, Isfahan
University of Medical Sciences, Isfahan, Iran
2Department of Microbiology and Virology, School of
Medicine, Kerman University of Medical Sciences, Kerman, Iran
3Department of Ophthalmology, Isfahan Eye Research Center,
Isfahan University of Medical Sciences, Isfahan, Iran
Corresponding author: Jamshid Faghri, Ph.D
Department
of Microbiology, School of Medicine, Isfahan University of Medical Sciences,
Isfahan, Iran
Tel/Fax:
+98 31 37929038/+98 31 6688597; Email: faghri@med.mui.ac.ir; http://orcid.org/0000-0001-7500-168X
Received:
October, 24, 2020; Accepted: December, 17, 2020
Abstract
Dacryocystitis is a lacrimal sac and duct inflammation.
It can be inherited or congenital. Two primary forms are acquired
dacryocystitis, acute and chronic. The goal of this study is to recognize
common bacteria causing nasolacrimal duct infection in children with congenital
nasolacrimal duct obstruction and to determine their antimicrobial
susceptibility profiles. This cross-sectional research was conducted from
January to February 2017 in the Department of Ophthalmology affiliated to Isfahan
University of Medical Sciences (center of Iran). Using phenotypic and genotypic
approaches, identification of specimens was performed. Disc diffusion method was
used for checking antibiotic susceptibility. All of the 59 isolates from the culture
of specimens belonged to Gram-positive cocci. Staphylococcus epidermidis
was the predominant species (n= 44, 74.6%) followed by Staphylococcus aureus
(n= 11, 18.6%), Staphylococcus haemolyticus (n= 2, 3.4%) and each of Staphylococcus
saprophyticus, and Streptococcus pneumoniae (n= 1, 1.7%). Totally, the
highest resistance was found against erythromycin and tetracycline while,
chloramphenicol, and ciprofloxacin showed the highest susceptibility. The
current research is useful in evaluating the suitable antibiotic in our area
for the systemic treatment of dacryocystitis. The most effective agents against
the most common isolates were chloramphenicol and ciprofloxacin. Since the
bacteriology of nasolacrimal duct infections varies from region to region, it
is recommended that further studies in other areas of our country can be
detected the etiology of bacterial pathogens involved in acute infections.
Keywords: Dacryocystitis, Antibiotic
resistance, Congenital nasolacrimal duct obstruction, Bacteriology
1.
Introduction
Dacryocystitis is an
inflammation of the lacrimal sac, usually followed by a blockage of the
nasolacrimal duct [1]. Dacryocystitis can be seen both in
acute and chronic forms. Acute type of dacryocystitis is an acute inflammation of
the lacrimal sac, the most important clinical symptoms of which include
discomfort, redness and swelling, and can be seen in 23 percent of cases of
lacrimal abscess [2, 3]. The chronic form of dacryocystitis
is more common than the acute form and is frequently associated with
conjunctivitis [4].
Approximately 60-90% of
all cases of lacrimal sac infection are related to bacterial dacryocystitis [5]. The microbial spectrum of the
dacryocyst depends on its acute or chronic form. In most cases, Gram-positive
bacteria are separated from acute dacryocystitis, while in the chronic form,
Gram-negative bacteria are predominant. The most important species isolated
from children include Staphylococcus aureus, Staphylococcus
haemolyticus, Streptococcus pneumoniae, and Haemophilus
influenzae, while Staphylococcus epidermidis, S. aureus, S.
pneumoniae, and Pseudomonas aeruginosa are the most causative agents
in adults [6]. Oral antibiotics,
anti-inflammatory drugs and local hot compresses were used in acute
dacryocystitis therapy methods, whereas definitive treatment of chronic dacryocystitis
is done with dacryocystorhinostomy [7].
The study of the status of drug resistance in dacryocystitis
is important in two aspects: first, according to studies, approximately
one-third of bacteria isolated are resistant to antibiotics, and on the other
hand, because in most cases, the treatment of patients is empirically and
without culture data if treatment failure results in consequences such as
cellulitis, meningitis, abscess, and even life-threatening conditions. In addition, the pattern of drug resistance varies in
different regions [8]. Early identification of microbial agents and awareness
of the drug susceptibility pattern is important for effective treatment. The
choice of antibiotic therapy for dacryocystitis usually depends on the age of
the patient, the condition of the patient (acute or chronic), the type of
microorganism and drug present, and the drug resistance pattern. Therefore,
epidemiologic studies are necessary for identifying and managing cases of
bacterial ýdacryocystitis [5, 6].
Since the range of
bacteriology may differ by geographical region, and very few studies have been
recorded from Central Iran on lacrimal sac bacteriology, the present study was
aimed at identifying bacteria involved in acute dacryocystitis in a given
population and investigating the trend of antibiotic susceptibility in the
province of Isfahan.
2.
Materials and Methods
2.1 Study
design, period, and area
A cross-sectional study
was conducted from January to February 2017 among dacryocystitis diagnosed
patients attending at Ophthalmology Outpatient of Feiz teaching hospital,
Center of Iran.
2.2 Inclusion/exclusion criteria
All children aged 6 years with a
history of dacryocystitis were referred to the selected clinic of ophthalmology
for sampling and 59 dacryocystitis cases were eligible for microbiological
analysis. Also, children with severe lid irritation due to
persistent discharge were included in this study. However, people over 6 years
old, the patients with the above symptoms who had received either topical or
systemic antibiotics for the past week and all
cases of canalicular obstruction were excluded from the study.
2.3 Sampling
After
aseptically cleaning the surrounding area, specimens for microbiological
analysis were obtained by sterile dacron swabs from the lacrimal sac, by applying
pressure over the lacrimal sac and allowing the purulent material to reflux
through the lacrimal punctum. The specimens were collected with the help of an
ophthalmologist and sent for microbiological analysis.
2.4 Microbiological analysis and bacterial identification
Specimens were inoculated
on BHI broth, chocolate agar, and blood agar (Oxiod, Hampshire, UK). Then, the inoculated media were incubated at
35-37 ºC for 24 to 48 hours. In addition, in the presence of 5-10 % carbon
dioxide, chocolate plates were incubated at 37 ºC for 24 to 48 hours.
Identification of the isolates was performed using different biochemical as
well as standard microbiological tests. To identify Gram-positive isolates
Standard biochemical and microbiological tests were included in order
(Catalase, Coagulase, PYR,
optochin susceptibility, etc.). The oxidase, novobiocin, and bacitracin tests
were used for the identification of coagulase-negative staphylococci.
Novobiocin disc is used to differentiate Staphylococcus saprophyticus from
other coagulase-negative staphylococci.
2.5
Antibiotic susceptibility pattern
Antibiotic susceptibility
pattern was performed by the disc diffusion method based on the Clinical and
Laboratory Standards Institute (CLSI) recommendation [9]. The following antibiotic disks were used;
ampicillin (10 μg), chloramphenicol (30 μg), gentamicin (10 μg),
tetracycline (30 μg), amoxicillin (20 μg), ciprofloxacin (30
μg), ceftriaxone (30 μg), erythromycin (15 μg). Also, Escherichia coli (ATCC 25922) and S.
aureus (ATCC 25923) were used as control strains.
2.6 DNA extraction and
Polymerase chain reaction (PCR)
The DNA template was
prepared as per the method of Dilhari et al. with slight alteration [10]. 200 μl
of phosphorus buffer saline (PBS) was taken in microcentrifuge tubes and a
loopful of each isolate was mixed with the nuclease-free water thoroughly in
each microcentrifuge tube. The suspended isolates in microcentrifuge tubes were
then treated with boiling water for 15 minutes. After heat treatment,
centrifuge tubes were centrifuged at 10,000 rpm for 10 minutes. 100 μl of
the supernatant containing genomic DNA transfer in a new tube and it was used
for subsequent PCR amplification. In our study, isolates were screened for the
presence of the 16s rRNA, femA, and Se705 genes. The sequences of primers
as shown in Table 1. The PCR reactions were conducted in
a total volume of 25 μL containing the following: 12.5 μl
of Master Mix (Ampliqon, Denmark), 11 μl distilled water, 1 μl
template DNA and 0.5μl primers. PCR
assay was performed in a DNA Thermal Cycler 480 (Applied Biosystems, USA). For
amplification femA, and Se705 genes, the PCR
program was set at:
denaturation for at 5 minute 94 °C, 29 cycles of 94 °C for 30 seconds, 53 °C
for 30 seconds, 72 °C for 30 seconds and a final extension step of 72 °C for 5
minutes.
Amplification of 16s rRNA according
to the following program: initial activation of 94 °C for 5 min, 35 cycles of
94 °C for1 min, 56 °C for1 min and 72 °C for 1 min and a final extension of 72
°C/7 min. Each
amplification reaction included a negative control (no-DNA template control). Five
microliters of the amplified DNA products were run on 1% agarose gel with 1 X TAE
(Tris/Acetate/EDTA) buffer, stained with safe stain load dye (CinnaGen Co.,
Iran) and visualized under ultraviolet illumination.
2.7 Data
analysis
The data
were entered and analyzed using SPSS version 16 statistical software program. A
p-value <0.05 was considered as a significant association between the
variables which were tested.
3.
Results
3.1 Patients’ demography
In the present study, 59
clinically diagnosed patients of dacryocystitis in children and both sexes were
studied over a period of two months. The average age of the patients was
studied 3 years (range <1–6 years), with a males’ predominance 57.6% (n=34)
compared to female 42.4% (n=25) with male and female ratio 34:25. Although, a
high frequency of dacryocystitis was reported in males than females, a
significant difference between males and females (P= 0.36) was not observed.
The most common age groups were the group under 1-2 years (33.9%) and the
lowest age group was 5 years (5.1%).
3.2
Bacterial isolates
From 59
positive samples, five different species were isolated. As
shown in Table 2,
S. epidermidis was the most commonly isolated organism followed by S.
aureus, then S. hemolyticous. S.
saprophyticus, and S. pneumoniae only detected one isolate
respectively.
3.3
Antimicrobial susceptibility test
The most effective
antibiotics against all organisms were chloramphenicol, and ciprofloxacin, each
showing affectivity of 100% (Table 3),
followed by cefoxitin and gentamycin with 61% and 49.7% while the highest
rate of resistance seen was to erythromycin, amikacin, and tetracycline, with
at least one resistant organism present in 40 of 59 (67.8%) cultures and 30 of
59 (50.8%), and 30 of 59 (50.8%), respectively.
3.4
Sequencing
Staphylococcus
hominis strain EFS 16S ribosomal RNA gene, partial sequence gene
with accession number “MG786536.1” were added in the database.
Discussion
This prospective study of
59 patients was done to determine the type of bacteria involved in patients
with acute dacryocystitis. The spectrum and incidence of pathogens, as well as
our study, were so obvious that we could isolate five different species of bacteria.
Microbial ocular surface flora consists predominantly of Gram-positive
microorganisms, namely staphylococci and diphteroids [12]. The
occurrence and severity of dacryocystitis depend on various factors, such as
the geography of the area and the type of microbial agent [10].
Badhu et al. (2006)
revealed that in Nepal the most common microorganism was S. pneumoniae,
while in some countries like Saudi Arabia, China, Austriaý, and Australia, S. ýepidermidis and S. aureus are the most frequent species
isolated from dacrysistis [13-17].
In the current study, the
most common organism isolated was S. epidermidis and S. aureus (74.6% and 18.6%,
respectively) this result is comparable
with other studies [1, 6, 17, 18].
However, this finding was much lower than the prevalence of Gram-positive
bacteria in dacryocystitis recorded in Saudi Arabia (79.1 %), Finland (62 %),
USA (68.8 %), Australia (54.4%), and in a previous study in Iran (68.8 %) [19-23]. In general, the most significant factors for
differentiating the diversity of bacteria in different studies include
variations in the number of patients, differences in the social status of
patients, access to ophthalmology, and public health knowledge among
individuals [6].
Operational intervention
is the main treatment of nasolacrimal duct obstruction. Despite surgical
procedures, the risk of infection of soft tissue increases fivefold without the
use of antibiotics, suggesting the importance of antibiotics in the treatment
of dacryocystitis [23]. On the other hand, resistance against
antibiotics is a problem that affects the treatment of dacryocystitis. In
recent decades, drug resistance has spread exponentially, which may be due to
overuse and abuse of these medications [24]. In our study, Gram-positive organisms
exhibited a high rate of sensitivity to chloramphenicol, vancomycin, and
ciprofloxacin. This is in correlation with the studies of Kuchar et al.
(2000), Kebede et al. (2010), Chung et al. (2019) also have
documented effectiveness of chloramphenicol, and ciprofloxacin against
Gram-positive bacteria [8, 23, 25, 26].
While S. epidermidis
and S. aureus displayed the highest susceptibility to chloramphenicol
and vancomycin, it should be noted that between 32% and 39% of antibiotic
resistance to these two drugs has been recorded. Therefore the determination of
bacterial species and the determination of drug sensitivity in patients with
dacryocystitis appear to be significant [6].
In a study by Assefa et
al. (2015) in Northwest Ethiopia, they found that the most susceptible
antibiotics were nalidixic acid (87.1%), erythromycin (84.2%), ceftriaxone
(95.3%), and gentamicin (83.3%) [18]. Briscoe et al. (2005) showed obtained
bacteria have ýmore sensitivity to ceftazidime
ýý(95%), ciprofloxacin
(86%), and cefuroxime (50%), ýrespectively
[27]. In the study by Shah et al. (2011)
norfloxacin was the most effective ýantibiotic while penicillin showed the most resistant
antibiotic [28]. The disparity between
studies shows that because of the regional pathogens, there are obvious
differences in the pattern of antibiotic resistance in the geographic areas [29].
The small sample size, and
short period, were the limitations of this research. A larger research group
with a longer study duration may provide a better result. Based on the results
obtained, we have offered to direct alternatives for the selection of effective
antibiotics for clinicians who take care of cases controlled for acute
dacryocystitis diagnosis.
The most frequent bacteria
isolated from acute dacryocystitis were S. epidermidis and S. aureus
in our area. The most effective antibiotics against all isolated microorganisms
from acute dacryocystitis include ciprofloxacin and chloramphenicol. These
'regional' results have significant public health consequences in this area of
Iran for the treatment and prevention of dacryocystitis.
Acknowledgment
We are grateful to the
study participants and Ophthalmology Department and Hospital Laboratory Staffs
of Feiz hospital for their help during data collection and sample processing.
Author
Contributions
HE, and JF, study
designed. AZ, and HE, carried out the experiments. HE, wrote the manuscript.
FS, analyzed the results. JF, supervised the project. HE, FS, and, AZ, edited
manuscript, submitted to the journal, and response to reviewers. All authors
read and approved the final manuscript.
Conflict
of Interest
None declared.
Ethical
declarations
This study has the formal
approval of the Research Ethics Committee of Isfahan University of Medical
Sciences, Isfahan, Iran (Approval number: IR.MUI.REC.1396.3.203).
Financial
Support
This work was supported by
the Isfahan University of Medical Sciences, Isfahan, Iran (Thesis code:
396203).
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Clinical and Laboratory Standards Institute (CLSI). Performance Standards for
Antimicrobial Susceptibility Testing; 27th ed. CLSI supplement M100. Wayne, PA:
Clinical and Laboratory Standards Institute; 2017.
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