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Clinical Microbiology & Case Reports
ISSN: 2369-2111
Research Aticle
Biofilm Formation and Presence of Esp and cylA Genes Enterococcus
faecalis Isolated from Hospital Infection
This article was published in the following Scient Open Access Journal:
Clinical Microbiology & Case Reports
Received May 20, 2015; Accepted June 01, 2015; Published June 09, 2015
Fariba Gapeleh1, Mohammad Reza Mehrabi2*,
Mohsen Mirzaee2 and Maryam Labibzadeh1
Department of Microbiology, Borujerd Branch,
Islamic Azad University, Borujerd, Iran
2
Assistant Professor Department of Laboratory
Sciences, Borujerd Branch, Islamic Azad University,
Borujerd, Iran
1
Abstract
Background and purpose: Enterococcus faecalis normal intestinal flora of humans
and one of the menangeditise the ability to form biofilm on surfaces such as catheters,
venous catheters artificial heart valves and ocular lenses, the ESP and cylA virolanse
factors in E. faecalis. The purpose of this study was to evaluate the ability of the bacteria
in the biofilm formation and detection of virulence factors in clinical isolates of enterococci
surface protein and cytolysin.
Method and Materials: A total of 54 clinical E. faecalis isolates was collected from
hospitals Ability of biofilm formation was measured by Microtiter plate assay. All isolates
were then examined for presence of the Esp and cylA genes by PCR.
Results: The microtiter plate assay results showed that attachment abilities in 4 (7%)
strains were strong, 10 (26%) strains were moderate, and in 14 (56%) strains were weak
and 5 (11%) strains didn’t form biofilm. The prevalence of the ESP and cylA genes identified
by PCR among clinical isolated strains, and the results were 83% and 70%, respectively.
Conclusion: Our results suggest different biofilm formation ability in clinical isolated
strains of E. faecalis. Biofilm formation on medical devices such as intravascular
catheters, artificial heart valves and ocular lenses can increase antibiotic resistance and
cause many health problems.
Introduction
*Corresponding authors: Mohammad Reza
Mehrabi, Department of Laboratory Sciences,
Borujerd Branch, Islamic Azad University, Borujerd, Iran,
Tel: 09166623598, Email: [email protected]
Volume 1 • Issue 3 • 018
Enterococcus faecalis Gram-positive cocci and natural inhabitant of the human
is the most common bacterial pathogens worldwide [1]. The third most common
cause of nosocomial bacteria is then Staphylococcus aureus and Escherichia coli [2].
Of the causes endocarditis, meningitis, urinary tract infections- endophthalmitis and
antibiotic resistance is extensive [3]. The emergence of antibiotic resistance threatens
the successful treatment of various infections [2]. It is the third leading cause of
infection after abdominal surgery and trauma caused by the removal of the epithelial
layer continuity and colon cancer [4]. Biofilms are complex microbial cell surface
polysaccharide matrix that binds irreversibly causing is bacteria survive in unfavorable
conditions [5]. The biofilm formation in bacteria is a social behavior that it passes over
three decades of research Enterococcus infections in the host’s ability to form biofilms,
biofilms cause survival stability and continuity in medical devices such as catheters prosthetics - implants and trauma treatment and medical cost increases. In addition to
the tolerance of the host immune system, such as phagocytosis cause genetic exchange
between cells forming the biofilm is very fast [6]. With increasing drug resistance in
enterococci studying virulence factors associated with colonization and pathogenesis
of this bacteria is essential Expression of specific genes and environmental conditions
with close ties to the biofilm formation of E. bonding on surfaces of medical tools such
as catheters - catheter and ocular lenses and the production of biofilm reported [7].
Enterococcus faecalis virulence factor of several hydrolytic enzymes, Surface proteins
and toxins [8]. ESP: This gene was first identified in MMH594 strains with1873 amino
acid and 202 kDa, 13 sequence conservation (744-1665) which N-terminal from (50743), which is essential in the action against the host. The c terminal (1666-1873),
which is located in the hydrophobic membrane [9] (Figure 1).
The presence of this gene is associated with urinary tract infections, bacteremia
[10]. Tendolkar and preeti in research found the presence of this factor in the ability
to form biofilms and biofilm thickness is proved. Removal of N-terminal region of the
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Clin Microbiol Case Rep
Citation: Gapeleh F, Mehrabi MR, Mirzaee M, Labibzadeh M (2015). Biofilm Formation and Presence of Esp and cylA Genes Enterococcus faecalis
Isolated from Hospital Infection
virulence factor in reducing the ability of bacteria to form biofilms
was impressive [11].
cylA: Part of an eight-subunit operon is the gene for the
removal of the N-terminal amino acid subunits and activation is
essential L-S subunit [4] (Figure 2).
Virulence factor in causing the uncontrolled release of
inflammatory mediators from damaged tissue and cells are
phagocytic. It is the production of extracellular enzymes and
toxins. Cytolysin moved on Mobile genetic elements such as
plasmids and exacerbated destruction of blood cells, Further
access pathogen to food and increased infection [12].
Biofilm Formation Assay
A modified microtiter plate method was followed as previously
described [13]. Briefly, the wells of microtiter plate were filled
with 180 μl of trypticase soy broth (TSB) supplemented with .5%
glucose. Then, a 20 μl quantity of previously prepared bacterial
suspensions with turbidity equal to 0.5 Macfarland standards
was added to each well. The negative control wells contained 200
μl of TSB supplemented with .5% glucose. Incubation was carried
Page 2 of 4
out at 37°C for 24 h before removal of the cultures. Then, the cells
were decanted, and each well was washed 3-times with sterile
phosphate buffered saline, fixed by methanol for 20 min, dried
at room temperature and finally strained with 0.1% saferanin.
The safranin dye bound to the adherent cells was dissolved with
1 mL of 95% ethanol per well, and the plates were read at 490
nm (A490) using ELISA reader. Optical density cut-off (ODc)
was determined. It is defined as average OD of negative control
+ 3× standard deviation (SD) of negative control. Formation of
biofilm by isolates was analyzed and categorized relying on the
absorbance of the safranin-stained attached cells (Table 1).
PCR screening for virulence-related genes: Genomic DNA
was extracted from pure cultures using a Bacterial Genomic DNA
Extraction Kit (cinapureTMDNAKIT, iran) and PCR was used to
detect the presence of the virulence determinants esp and cylA.
The primer sequences were blast NCBI site and synthesized by
pishgam biotechnology company and PCR procedures were set
based related references (Table 2).
Cut-off value calculation
Mean of OD values
results
OD > 4×ODc
Biofilm formation
abilities
OD >.216
Strong
2×ODc < OD ≤4×ODc
.108< OD ≤.216
Moderate
ODc< OD ≤ 2×ODc
.054< OD ≤.108
Weak
OD ≤ .054
None
OD ≤ .054
Table 1: Classification of biofilm formation abilities by Mtp method.
Reference Product Size Programe
(bp)
PCR
[15]
[5]
Figure 1: Gene ESP.
Sequence
Target
(s)
95
60s 95°C,
60 s 63°C,
60 s 72°C
114
60s 95°C,
F-TTATGCATCAGATCTCTCAA
60 s 58°C,
cylA
R-CCGAGTGCTTGCACTCAATTGG
60 s 72°C
F-GAACGCCTTGGTATGCTAAC
R-CCACTTTATCAGCCTGAACC
ESP
Table 2. Primer sequences and program used in PCR.
Figure 2: Gene cylA.
Volume 1 • Issue 3 • 018
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Clin Microbiol Case Rep
Citation: Gapeleh F, Mehrabi MR, Mirzaee M, Labibzadeh M (2015). Biofilm Formation and Presence of Esp and cylA Genes Enterococcus faecalis
Isolated from Hospital Infection
54 isolates E. faecalis were selected for molecular screening
for esp and cylAgenes using PCR.
PCR amplification was performed with an Ep¬pendorf
thermal cycler (BIO RAD). Amplification program for ESP
consisted of initial denaturation at 95°C for 5 min, 35 cycles of
denaturation at 95°C for 60 sec, annealing at 63°C for 60 sec and
extension at 72°C for 60 sec with a final step of 72°C for 10 min.
The PCR products were analyzed by electrophoresis in a 2%
agarose gel and stained with gel red.
cylA gene detection
Amplification program for cylA consisted of initial
denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C
for 60 sec, annealing at 58°C for 60 sec and extension at 72°C for
60 sec with a final step of 72°C for 10 min (Figure 3).
Clinical bacterial strains
The microtiter plate assay results showed that attachment
abilities in 4 (7%) strains were strong, oundes in 10 (26%) strains
were moderate, and in 14 (56%) strains were weak and 5 (11%)
50bp
Figure 3: The presence of the esp and cylA genes in clinical isolates of
Enterococcus faecalis using PCR.
BIOFILM
M
26%
56%
N
S
11%
W
strains didn’t form biofilm. The prevalence of the ESP and cylA
genes identified by PCR among clinical isolated strains, and the
results were 83% and 70%, respectively (Figure 4 and Table 3).
Discussion
Enterococci are as the natural microflora of the intestinal
tract of humans and animals.
The bacteria under certain conditions lead to the emergence
of urogenital tract infection, inflammation of bile ducts,
endocarditis, meningitis and infections the skin. Several reports
suggest an increase in innate and acquired resistance of bacteria
and biofilm production is at least 16 the epidemic caused by multiresistant enterococci have been reported from 1989 to 1998 [11]).
Toledo and Arena to study the expression (P<.0001) estimated
of the relationship between the presence of Espgene and biofilm
formation on the surface of Polystyrene. Biofilm formation in
urine collection bag associated with the presence of the Esp gene
[2]. Darini and colleagues in Brazil ESP genes effective in Biofilm
formation and increases antibiotic resistance [14]. Shankar et
al. stated that the N-terminal region of the gene Esp structural
changes in the bacterial will help organism’s ability escape from
the host immune response [15]. Chinorose and colleagues stated
that the prevalence of ESPgene among strains resistant to highly
gentamicin (86%) is associated [9]. Van Dyne D and colleagues in
the study stated that the presence cytolisin gene is exacerbated
of infection in humans [1]. Coburn and his colleagues in the study
stated is related to the expression of this operon cytolisin with
Biofilm formation and biofilm formation synergistic factor AS and
collaboration with Esp genes are highly interrelated [4]. Chinorose
and colleagues in a study of the prevalence of isolates resistant to
gentamicin cylA genes, 57 percent indicated this gene is associated
with biofilm production [9]. Our research was reported in the
gene esp 83% and the prevalence of the gene cylA 70% of the
geographical area and genetic characteristics of different strains
may be due to differences in prevalence. Enterococcal is second
cause bacteremia and endocardit and the third most common
cause of urinary tract infection and urinary tract infection and
bacteremia is the presence of the esp gene is associated. Given
the high prevalence of these genes among the isolates in the study
and collection of urine samples of both studies were consistent.
Enterococcus effective bonding on surfaces of medical tools such
as levels venous catheters - urinary and ophthalmic lenses and the
production of biofilm bacteria in biofilms are reported because
high concentrations of antibiotics tolerated. Appears at medical
centers play an important role in the pathogenesis of antibiotics
is the responsibility of Enterococcus. The use of antibiotics in
these disease-gene expression and increased levels of inducible
factors and pathogenesis of this bacteria increases.
Conclusion
7%
Figure 4: Diagram prevalence of biofilm.
esp negative
esppositive
cylA negative
No biofilm
1
5
1
5
Weak biofilm
7
23
11
19
Moderat biofilm
1
13
4
10
Stron biofilm
CylA positive
0
4
0
4
Table 3. The relationship between the genes and the level of biofilms.
Volume 1 • Issue 3 • 018
Page 3 of 4
Our results suggest different biofilm formation ability in
clinical isolated strains of E.faecalis. Biofilm formation on medical
devices such as intravascular catheters, artificial heart valves and
ocular lenses can increase antibiotic resistance and cause many
health problems.
We found relationships between biofilm formation and
prevalence of virulence genes esp and cylA. 89% of the strains
were able to form biofilm. The thickness of the biofilm increased
in the presence of these genes.
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Clin Microbiol Case Rep
Citation: Gapeleh F, Mehrabi MR, Mirzaee M, Labibzadeh M (2015). Biofilm Formation and Presence of Esp and cylA Genes Enterococcus faecalis
Isolated from Hospital Infection
Suggestions
Due to the presence of Enterococcus faecalis in hospitals,
especially in prolonged hospitalization to reduce biofilm
formation by bacteria and reduce antibiotic resistance can
observe the following:
1. Cleaning and sterilization of medical devices
2. The use of urinary catheters and intravenous disposable
3. Investigation personnel carrier in terms of Enterococcus
faecalis
4. Recognized source and possible routes of infection may
prevent the formation of biofilms are effective.
Acknowledgment
This article is part of the research work is graduate thesis.
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Page 4 of 4
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Copyright: © 2015 Fariba Gapeleh. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
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