This research aimed to investigate the antibacterial activity of Camphor extract ,against biofilm formation of clinical isolates Staphylococcus aureus and Staphylococcus lentus .by detecting the presence and absence of virulence genes that responsible for the biofilm formation after treatment  with Camphor  extract. .thirty isolates were diagnosed initially as Staphylococci then selected five isolates depending on resistance to different type of antibiotics as virulence .After that the isolates test by VITEK-2compact system (ID and AST) to confirm the species of Staphylococci .The diagnostic result showed that three isolates belong to Staphylococcus aureus and two to Staphylococcus lentus. Investigation of the ability of camphor oil to inhibit the growth of bacteria using diffusion method in Muller Hinton agar ,the results showed the effect of camphor oil to prevent biofilm formation  on both bacterial species with concentration (0.1 g/ml )

Jasim AL Kaabi, Siham

Mohammed Hussein AL Kinani, Roaa

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Al-Kufa University Journal for Biology                                     
                                           Print ISSN: 2073-8854                Online ISSN: 2311-6544 
University of Kufa / Faculty of Education for Girls 
Third International Scientific Conference 5-6/12 /2018 
62 
 
Detection  of Gene Involved in Biofilm Formation in Methecillin Resistance 
Staphylococcus aureus and Staphylococcus lentus and Effect of Camphor 
extract on These Genes 
Roaa Mohammed Hussein AL Kinani                             Siham Jasim AL Kaabi 
Biology of Department ,Faculty of Education for Women ,University of Kufa ,Iraq,  E-mail: 
mekesaif@gmail.com 
Abstracts 
   This research aimed to investigate the antibacterial activity of Camphor extract ,against 
biofilm formation of clinical isolates Staphylococcus aureus and Staphylococcus lentus .by 
detecting the presence and absence of virulence genes that responsible for the biofilm 
formation after treatment  with Camphor  extract. .thirty isolates were diagnosed initially as 
Staphylococci then selected five isolates depending on resistance to different type of 
antibiotics as virulence .After that the isolates test by VITEK-2compact system (ID and AST) 
to confirm the species of Staphylococci .The diagnostic result showed that three isolates 
belong to Staphylococcus aureus and two to Staphylococcus lentus. Investigation of the ability 
of camphor oil to inhibit the growth of bacteria using diffusion method in Muller Hinton agar 
,the results showed the effect of camphor oil to prevent biofilm formation  on both bacterial 
species with concentration (0.1 g/ml ). A molecular investigation of susceptibility of 
Bacteria to biofilm formation was also observe by the presence or absence of 
virulence gene that responsible of biofilm formation represented by icaA, icaD gene 
that responsible for intercellular adhesion by using PCR Technique. And using 
specialized primer, the results showed that all isolates of S.aureus contained both 
inherited and loss icaA in S.lentus isolated. This is evidence of the susceptibility of 
staphylococcus aureus for biofilm formation. 
 
Key words : camphor, biofilm ,methicillin ,Staphylococcus aureus , Staphylococcus 
lentus.icaA,icaD. 
 Introduction 
      The medicinal plants have been of great interest by researchers in the last few years, 
despite the development in the manufacture of medicines made of pure chemicals or 
biological materials such as antibiotics and given the effects of these substances on the public 
health of the individual, In addition to the increase in the number of micro-organisms resistant 
mostly for continuous use and without medical consultation (1). The extract of camphor is 
among the plants belonging to the family Eucalyptus, which has high pharmacological 
effectiveness because it is rich in essential oils,like cineole With the killer properties of 
microorganisms (2) In addition to the phenolic compounds that prevent the formation of 
biofilm impact on the process of Quorum sensing (QS) (3). We can define biofilm by a 
collection of bacteria attached to living and non-living surfaces, surrounded by substances 
secreted by bacterial groups, which are extracellular polymers (4),which is used to hide from 
the host's immune system and resistance to different type of antibiotics (5).Staphylococcus 
aureus and the Staphylococcus lentus caused many health problems including skin infections 
with deep inflammation and inflammation of the bone, chronic ostemyelitis ,endocarditis and 
other heart diseases that may lead to death  (6). 
  
Materials and Methods 
Collection and preparation of Camphor Oil extract 
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                                           Print ISSN: 2073-8854                Online ISSN: 2311-6544 
University of Kufa / Faculty of Education for Girls 
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63 
 
   Various forms of camphor were collected from the local markets of Najaf Governorate and 
the local markets of Baghdad governorate, using hard camphor, which is a crystalline cube 
from Deer brand/China,The camphor oil was prepared by dissolving 3 g of camphor in 30 ml 
of sterilized olive oil at 121 ° C and under pressure of 15 lb / kg
2
 for 15 minutes, in a water 
bath vibrating 95 ° C for 15 minutes until it was confirmed Melting the camphor completely 
with olive oil then placed in a sterile tube and kept in the refrigerator 4 ° C until it is used and 
the solution to the storage of pure camphor extract (7). 
Collection of specimens 
   A total 5 specimens which were collected from urinary  tract infection, wound, burns and 
swabs from the indoor environment in hospital of Najaf govermorate during a period ( from 
September 2017 to February 2018) , patient’s age ranged from ( 15 years - 35 years). The 
samples were processed on Blood agar medium ,Nutrient agar and Mannitol salt agar and 
were incubated at 37°C overnight. The identification of Gram positive bacteria, performed by 
standard biochemical methods (catalase test, oxidase test, Mannitol Fermentation ,and also 
diagnosed by using Vitek-2 compact system . 
Detection the inhibitory activity of camphor oil towards staphylococcus aureus and 
staphylococcus lentus 
   The well diffusion method was used to study the susceptibility of the camphor oil to the 
bacterial isolates(8). The isolated bacteria were spread homogenously on the Muller Hinton 
agar medium and left at room temperature to ensure that the bacterial implants in the center, 
Then, the center was drilled with a diameter of 8 mm and the camphor oil was added at a 
concentration of 0.1 g / ml. Excavation of positive control containing olive oil only worked 
separately to ensure that there was no inhibitory effect of olive oil on developing colonies. 
Leave the dishes for 10 minutes with room temperature to allow the extract to spread through 
the middle. It was incubated 37 ° C for 24 hours and the diameters of the drill were measured 
using a graduated ruler. 
DNA template preparation by using boiling method with Tris EDTA-TE buffer : 
   The total DNA of isolates under study was extracted using the Tris EDTA-TE Buffer 
method (9) With some modifications made by the researcher to suit the search requirements:, 
grown Bacterial isolates in BHI Broth at 37 ° C for 24 h.then discarded centrally after placing 
the tubes in the cannula for 5 minutes at a speed of 5000 rpm. The precipitate dissolved in 1 µl 
of TE Buffer ,then   The tubes were centrifuged centrally at 5000 rpm for 5 minutes, after that 
re-suspended The percipitate in100 µl of TE Buffer to ensure that all the cells are absorbed, 
The tubes were placed in a 100 cº water bath for 10 minutes and immediately discarded for 5 
minutes at 5000 rpm,.then Collect the supernatant  from the tube and keep in freezing at -20 ° 
C until use.  
Primers used for PCR 
   The genes used in this study are responsible for intercellular adhesion necessary for Biofilm  
formation of Staphylococcus aureus obtained from the Korean Alpha DNA Company as 
shown in the table1. 
 
 
 
 
Table -1:The primer used in the current study for PCR amplification. 
       
 Product Nucleotide sequences and direction(5
\
-----3
\ (  Target 
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University of Kufa / Faculty of Education for Girls 
Third International Scientific Conference 5-6/12 /2018 
64 
 
 
Reference 
 
size (bp) Gene 
(10) 151 
5
\  
GAG GTA AAG CCA ACG 
CAC TC 3
\
 
F Ica A 
5
\
 CCT GTA ACC GCA CCA AGT 
TT 3
\
 
R Ica A 
(11) 483 
5
\ 
GAA CCG CTT GCC ATG TGT 
TG 3
\
 
F Ica D 
5
\
 GCT TGA CCA TGT TGC GTA 
ACC 3
\ 
 
R Ica D 
 
PCR amplification procedure                                                                          
   Five selected isolates were submitted to genotypic study using PCR.The oligonucleotide 
primers for the icaA and icaD genes (table 1) were diluted using TE Buffer according to the 
manufacture company information (Alpha DNA) to get primary concentration equal to 100 µl 
.The amplification was performed in PCR thermocycler  and the reaction mixture was 
prepared according to the procedure that suggested  by the manufacture company (Maxime 
PCR PreMix kit (i-Taq)). The components of the polymerase chain reaction are shown in 
the following table 2 
Table 2: component and volumes of  PCR  mixture 
Volume PCR Master Mix 
10 µl DNA Template 
4 µl Forward primer  
4 µl Reverse primer 
2 µl Nuclease free water 
20 µl Total 
Conditions for PCR amplification which were set according to this study for the two primers 
was shown in table 3 
Table 3 : Program of thermal cycles to amplify DNA 
 
Cycle 
No. 
Temprature (C) /Time                             Gene 
Final 
Extention 
Cycling Condition             Initial 
denaturation Extention Annealing denaturation 
 30 72/7 min 72/2 min 55/1 min 94 / 1 min 94/2 min Ica A 
30 72/7 min 72/2 min 60/1 min 94/1 min 94/2 min Ica A 
30 72/7 min 72/2 min 57.5/1 min 94/1 min 94/2 min Ica D 
30 72/7 min 72/2 min 60/1 min 94/1 min 94/2 min Ica D 
  
        The amplified PCR  product were analyzed by agarose gel electrophoresis by using 1.5 
% agarose supplied with 0.5 µg/ml ethidium bromide for 1 hour(12) .DNA ladder (100-1500 
bp) were used to assess PCR products were visualized by UV light at 336 nm ,and 
photographs were taken using digital camera. 
 
Results and Discussion 
      Spread of methicillin –resistance Staphylococcus aureus  and Staphylococcus lentus is an 
important concern in hospitals and other health care .a total of 5 specimens showed  that 3 
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                                           Print ISSN: 2073-8854                Online ISSN: 2311-6544 
University of Kufa / Faculty of Education for Girls 
Third International Scientific Conference 5-6/12 /2018 
65 
 
isolates belong to Staphylococcus aureus and 2 to Staphylococcus lentus  according to 
different biochemical test including culturing on Mannitol salt agar and here S.aureus isolates 
able to converting this media color from red to yellow  due to Mannitol fermentation, 
Coagulase test also used as remarkable diagnostic test for S.aureus that has the ability to give 
positive result (13),Another important diagnostic test was performed using Vitek-2 compact 
system To confirm the diagnosis (14) this two species differ according to source of the 
isolates ,nature , the Age of the patient ,and geographic location that samples were taken from 
.By using diffusion method to detect the antimicrobial activity of Camphor oil (0.1 g/ml) ,it 
was 40mm in diameter  for  S. aureus isolates and 44mm in diameter for S.lentus isolates as 
shown in the following figure1 
 
 
 
Figure1: shows the anti-microbial effect of camphor oil towards the isolation of S.aureus on 
Muller Hinton agar at concentrated(0.1 g/ml)  
       The inhibitory efficacy of oil is likely to be due to its active ingredients, notably phenols 
In addition to volatile oils, especially Cineole oil, which constitutes about 80-90% one of the 
total active substances of the plant ,Which works to destroy the cell membrane and destroy the 
bacterial cells (15).then this five isolates were selected for the genotypic study by PCR 
technique using two different gene sequence Which are important in the process of biofilms 
formation and it is ica gene (inter cellular adhesion ) which responsible for adhesion between 
cell during biofilm formation and it’s the first step in this process ,the first one is icaA with 
amplified size equal to 151 bp(10) and the second is icaD with amplified size equal to 483 
bp(11).The result of PCR experiment revealed that the possession of isolates (S1 
aureus,S3aureus) icaA gene with anneling tempreture 55 cº ,while the isolates 
(S1aureus,S2aureus ,S3aureus) possessing icaA gene with anneling tempreture 60 cº ,but did 
not show the isolates (S4 lentus,S5 lentus) the gene in the same temperature as in the figure 2 
                   
 
  
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                                           Print ISSN: 2073-8854                Online ISSN: 2311-6544 
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                    60 cº                              50 cº 
 
Figure 2:Agarose gel electrophoresis (1.5% agarose ) of icaA gene (S1,S2,S3)is S.aureus 
isolates (,S4,S5) is S.lentus isolates ,S6 is control 
    While the result of icaD gene showed that all isolates possessing the gene with two 
annealing temperature   (57.5-60)cº as in figure 3        
                                                       60 cº                                                     57.5cº    
 
Figure3: Agarose gel electrophoresis (1.5% agarose ) for icaD gene (S1,S2,S3)is S.aureus 
isolates (,S4,S5) is S.lentus isolates ,S6 is control 
            The presence an absence of virulence genes in the isolates may be due to the sources 
of collection of samples ,as the use of detergents and disinfectants in hospitals can lead to a 
change in the nature of genetic materials constituent of the cells and the disappearance of 
some these genes (17). 
 
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University of Kufa / Faculty of Education for Girls 
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Detection  of Gene Involved in Biofilm Formation in Methecillin Resistance Staphylococcus aureus and Staphylococcus lentus and Effect of Camphor extract on These Genes

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Detection of Gene Involved in Biofilm Formation in Methecillin Resistance Staphylococcus aureus and Staphylococcus lentus and Effect of Camphor extract on These Genes

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