Detection of the intercellular adhesion gene cluster (ica) in clinical Staphylococcus aureus isolates

dgkh000203 10.3205/dgkh000203 urn:nbn:de:0183-dgkh0002035 Research Article Detection of the intercellular adhesion gene cluster (ica) in clinical Staphylococcus aureus isolates Nachweis des interzellulären Adhäsions-Gencluster (ica) in klinischen Staphylococcus aureus-Isolaten Namvar Namvar Amirmorteza Ebrahimzadeh AE

Medical Microbiology Department, Antimicrobial Resistance Research Center, Faculty of Medicine, Iran University of Medical Science, Tehran, Iran

author Asghari Asghari Babak B

Medical Microbiology Department, Antimicrobial Resistance Research Center, Faculty of Medicine, Iran University of Medical Science, Tehran, Iran

author Ezzatifar Ezzatifar Fatemeh F

Medical Microbiology Department, Antimicrobial Resistance Research Center, Faculty of Medicine, Iran University of Medical Science, Tehran, Iran

author Azizi Azizi Gholamreza G

Imam Hassan Mojtaba Hospital, Iran University of Medical Sciences, Tehran, Iran

author Lari Lari Abdolaziz Rastegar AR

Medical Microbiology Department, Antimicrobial Resistance Research Center, Faculty of Medicine, Iran University of Medical Science, Tehran, Iran, Phone: +98218293181Medical Microbiology Department, Antimicrobial Resistance Research Center, Faculty of Medicine, Iran University of Medical Science, Tehran, Iran

lari@tums.ac.ir author German Medical Science GMS Publishing House

Düsseldorf

610 Staphylococcus aureus biofilm intercellular adhesion gene PCR detection Staphylococcus aureus Biofilm Interzelluläres Adhäsionsgene PCR Detektion Adhäsionsfaktor 2013042920130710 engl 2196-5226 8 1 GMS Hygiene and Infection Control GMS Hyg Infect Control 03 20130710The ISSN has been corrected. Staphylococcus aureus ist ein wichtiger nosokomialer und community-assoziierter Krankheitserreger, der verschiedene humane Infektionen verursachen kann. Durch die Fähigkeit von Mikroorganismen zur Biofilmbildung wird ihre Widerstandsfähigkeit gegenüber der Immunabwehr mit der Folge chronischer oder persistierender Infektionen erhöht. Es wurde nachgewiesen, dass durch ica-codierende Gene die Biosynthese des interzellulären Polysaccharid-Adhäsins exprimiert wird, das eine Hauptkomponente für die Akkumulation des Biofilms darstellt. In verschiedenen Studien wurde die kritische Rolle der ica-Gene als Virulenzfaktor für Staphylokokken-Infektionen nachgewiesen.Die vorliegende Untersuchung wurde durchgeführt, um den Zusammenhang zwischen dem ica- Gen und der Schleimbildung durch S. aureus zu analysieren. Hierzu wurden 60 S. aureus Patientenisolate identifiziert, morphologisch und biochemisch nach Standardmethoden charakterisiert und in Trypticase-Soja-Bouillon (TSB) mit Zusatz von 15% Glycerol bei –20°C aufbewahrt. Die Schleimbildung und Biofilmbildung wurde in einem speziellen Assay detektiert. Mittels eigens entwickelter PCR wurde alle Isolate auf das Vorkommen des icaD (intercellular adhesion gene) untersucht. Von den auf CRA-Platten Schleim produzierenden Kolonien waren 39 (65%) schwarz, die anderen 21 (35%) pinkfarben. Im quantitativen Biofilmassay bildeten 35 der Isolate (58%) einen Biofilm. Bei all diesen Isolaten wurde das icaD-Gen nachgewiesen. Des lässt den Schluss zu, dass der PCR-Nachweis des ica-Locus als rasche und effektive Methode zur Unterscheidung zwischen potentiell virulenten and avirulenten Isolaten herangezogen werden kann, um die Therapie und Prävention der Biofilmbildung auf Implantaten zu verbessern. Der Synergismus zwischen icaA und icaD in den untersuchten Isolaten scheint bedeutend für die Schleimbildung und Biofilmakkumulation sein. Staphylococcus aureus is a major hospital and community pathogen having the aptitude to cause a wide variety of infections in men. The ability of microorganisms to produce biofilm facilitates them to withstand the host immune response and is recognized as one factor contributing to chronic or persistent infections. It was demonstrated that the ica-encoded genes lead to the biosynthesis of polysaccharide intercellular adhesion (PIA) molecules, and may be involved in the accumulation phase of biofilm formation. Different studies have shown the decisive role of the ica gene as virulence factors in staphylococcal infections. This study was carried out to demonstrate the relationship between ica gene and production of slime layer in S. aureus strains. Sixty S. aureus strains were isolated from patients. The isolates were identified morphologically and biochemically following standard laboratory methods. After identification, the staphylococcal isolates were maintained in trypticase soy broth (TSB), to which 15% glycerol was added, and stored at –20°C. Slime formation and biofilm assay was monitored. A PCR assay was developed to identify the presence of icaD (intercellular adhesion gene) gene in all isolates. Thirty-nine slime producing colonies with CRA plates (65%) formed black colors, the remaining 21 isolates were pink (35%). In the quantitative biofilm assay 35 (58%) produced biofilm while 25 (42%) isolates did not exhibit this property. All isolates were positive for detection of icaD gene by PCR method. The interaction of icaA and icaD in the investigated isolates may be important in slime layer formation and biofilm phenomena.We propose PCR detection of the ica gene locus as a rapid and effective method to be used for discrimination between potentially virulent and nonvirulent isolates, with implications for therapeutic and preventive measures pertainin to the management of colonized indwelling catheters. IntroductionStaphylococcus aureus is an important pathogen causing a wide spectum of infections , . A number of studies have been conducted to explain the structures and pathogenic mechanisms by which S. aureus is able to cause serious infections , . The ability of S. aureus to produce biofilm enables this organisms to withstand the host immune response and is considered to be the cause of chronic or persistent infections, as biofilm creation protects bacteria from opsonophagocytosis and antimicrobial agents . Another concern related to this pathogen is increasing resistance to oxacillin and other antibiotics, but also distribution of multiresistant isolates within the hospital setting . Staphylococcal pathogenesis is multifactorial, involving a combination of adherence and biofilm formation . Complex aggregations of microorganisms can form irreversible attachments to the surfaces and formation of biofilm . Biofilm producing bacteria are the source for persistent or chronic infections . The significance of biofilm production for the virulence of S. aureus was supported by a number of clinical and animal studies . Cell aggregation and biofilm accumulation are mediated by the products of a gene locus composing of the genes icaADB and C, which encode the essential proteins for the production of polysaccharide intercellular adhesion (PIA) and capsular polysaccharide/adhesion (PS/A) in Staphylococcus spp. , . It was demonstrated that the ica-encoded genes are responsible for the biosynthesis of the PIA, which contains N-acetylglucosamine as a main constituent and in the accumulation phase of biofilm formation, playing a crucial role in invasiveness of S. aureus . Different studies have shown the decisive role of the ica gene as virulence factors in staphylococcal infections , .In this study, we developed new primers specific for icaD and determined further the possible relationship between icaD gene and producing slime layer in clinical isolates of S. aureus strains. Materials and methods1. Bacterial isolates and phenotypic identificationSixty S. aureus strains were isolated from patients and used in this study. Isolates were identified morphologically and biochemically by standard laboratory methods. The coagulase and DNase tests were performed for discrimination of S. aureus from coagulase negative staphylococci (CoNS). After identification, the staphylococcal isolates were maintained in trypticase soy broth (TSB), to which 15% glycerol was added, and stored at –20°C.2. Slime forming colonyThis test was used to evaluate slime formation in S. aureus strains on CRA plates, as previously described . Pink colonies were recorded as nonbiofilm producers while the black colonies were recognized as biofilm producers. 3. Biofilm assayS. aureus strains were icubated in (TSB) at 37°C for 24 hours</PlainText></TextGroup>; grown colonies were diluted in 1:200 and incubated in microtiter plates. After 24 hours the wells were washed with PBS buffer two up to three times and left in the room temperature for drying. In the next step 0.4% crystal violet solution was used as stain for 10 min. Finally the absorbance at 490 nm was determined; an OD of 490 nm >0.12 was regarded as a biofilm positive sample. </Pgraph><SubHeadline>4. Primer design and PCR amplification</SubHeadline><Pgraph>PCR assay was achieved to detect the presence of <Mark2>icaD</Mark2> (intercellular adhesion) gene in all isolates. For preparing DNA, colonies of bacteria were dissolved in 20 µl lysis buffer (0.25% SDS, 0.05N NaOH) and heated at 95°C for 5 min. In the second step, the lysate was centrifuged and diluted using distilled water. A second centrifugation step for 5 min at 16,000 g was performed to remove the cell debris. Supernatants were frozen at –20°C until further use. The primer sequences <Mark1>F: 5' GAA CCG CTT GCC ATG TGT TG 3' (20 bp) & R: 5' GCT TGA CCA TGT TGC GTA ACC 3' (21 bp)</Mark1> for amplifying a 483 bp of <Mark2>icaD</Mark2> gene was designed from the published GenBank sequences (AF086783) with Alleleid 6 Primer Analysis Software. For recognition of <Mark2>icaD</Mark2> gene each 25 µl PCR mixture contained 2.5 µM of MgCI<Subscript>2</Subscript>, 100 µM of each dNTPs, 1U Taq DNA polymerase, and 1 µM of each primer with 200 ng of the DNA sample. PCR amplification was carried out with use of a DNA Thermal cycler (Eppendorf Master cycler personal) with an initial denaturation step (2 min at 95°C) followed by 30 cycles of amplification (denaturation at 95°C for 30 s, annealing at 57.5°C for 30 s and elongation at 72°C for 40 s). Steps 2–4 were repeated 29 more times. Finally, 7 µl of PCR mixture was analyzed by 1.5% agarose gel electrophoresis. After electrophoresis, gels were seen under UV light. The Gene Ruler TM 100 bp DNA Ladder Plus (Fermentas, Germany) was used as a DNA ladder.</Pgraph></TextBlock> <TextBlock linked="yes" name="Results"> <MainHeadline>Results</MainHeadline><Pgraph>The investigated 60 strains were isolated from samples of blood catheter (40%), urine catheter (27.5%), sputum (8.3%), wounds (10%), and tracheal tube (14.2%), during the period October 2011 to February 2012. Thirthy-nine slime producing colonies with CRA plates (65%) formed black colors and, the remaining 21 were pink (35%). In the quantitative biofilm assay 35 (58%) produced biofilm, while the remaining 25 (42%) isolated did not exhibit this ability. All of these isolates were positive for detection of<Mark2> icaD</Mark2> gene by PCR method (Figure 1 <ImgLink imgNo="1" imgType="figure"/>). </Pgraph></TextBlock> <TextBlock linked="yes" name="Discussion"> <MainHeadline>Discussion</MainHeadline><Pgraph><Mark2>S. aureus</Mark2> is responsible for infections in humans. It has been demonstrated that strains having an ability to form biofilm cause additionally “chronic polymer-associated” infection <TextLink reference="9"></TextLink>, <TextLink reference="12"></TextLink>, mostly een as implant-associated infections. Biofilm support the adhesion and colonization of <Mark2>S. aureus</Mark2> on surfaces, frequently leading to persistent and difficult to eradicate infections <TextLink reference="4"></TextLink>, <TextLink reference="7"></TextLink>. Indeed, an increasing number of different <Mark2>S. aureus</Mark2> adhesion mole<TextGroup><PlainText>cule</PlainText></TextGroup>s is found <TextLink reference="13"></TextLink>. The <Mark2>ica</Mark2> operon of <Mark2>S. aureus</Mark2> and <Mark2>S. epidermidis</Mark2> contain <Mark2>ica</Mark2> <Mark2>(ADBC)</Mark2> that allow <Mark2>Staphylococcus spp</Mark2>. to form slime layers and biofilm. It was shown that the presence of <Mark2>icaA</Mark2> gene in microorganisms yielded from indwelling catheter samples from patients hospitalized for one week was notably frequent. Cramton et al. <TextLink reference="14"></TextLink> demonstrated the presence of <Mark2>icaA </Mark2>gene in <Mark2>S. aureus</Mark2> strains, which was confirmed by Arciola et al. <TextLink reference="1"></TextLink> describing 23 <Mark2>S. aureus</Mark2> starins isolated from 14 catheter associated infections with the ability to form slime layer based on te presence of the gene <Mark2>icaA,</Mark2> but also <Mark2>icaD</Mark2>. Indeed, while<Mark2> icaA</Mark2> is required to encode N-acetylglucosaminyltransferase, coexpression of <Mark2>icaD</Mark2> can increase the capsular polysaccharide phenotypes <TextLink reference="15"></TextLink>, indicating a significant role of the <Mark2>icaD</Mark2> locus as a virulence factor in the pathogenesis of <Mark2>S. aureus</Mark2> isolated from catheters <TextLink reference="13"></TextLink>, <TextLink reference="16"></TextLink>.</Pgraph><Pgraph>Infections associated with the use of invasive medical devices, e.g., catheters, are mainly due to <Mark2>S. aureus</Mark2>, particularly those strains which create an extracellular slime and parts of the biofilm, making clinical treatment extremely challenging. The pocess of biofilm formation needs polysaccharide intercellular adhesion, which is synthesized by the enzymes encoded by the intercellular adhesion cluster (<Mark2>ica</Mark2>). In this study, the percentage of slime producing strains was 58%, while 65% of these had black colonies on CRA plate. However, all isolates contain <Mark2>icaD</Mark2> gene. Our study showed that <Mark2>S. aureus</Mark2> isolates had no ability to form biofilm unless they were positive for <Mark2>icaD</Mark2> gene. Subsequently, the PCR product was purified from the gel and sequenced (sequence accession no.: JN226155).</Pgraph><Pgraph>Since the presence of adhesion molecules is required for the establishement of an infection, the presence of ica adhesion genes may explain the role of the various adhesion mechanisms in the pathogenesis infection associated with in-dwelling medical devices <TextLink reference="17"></TextLink>, <TextLink reference="18"></TextLink>. It can be concluded that infections caused by <Mark2>ica</Mark2> locus carriing <Mark2>S. aureus</Mark2> strains can lead to clinically difficult to treat conditions. The detection of the<Mark2> ica</Mark2> locus in clinical <TextGroup><Mark2>S. aureus</Mark2></TextGroup> isolates may improve the clinical decision for treatment and prevention options, and could support development of strategies to interact the bacterial capacity to colonize and invade in-dwelling medical devices. PCR detection of the <Mark2>ica</Mark2> operon may be an effective method to differentiate between virulent and non virulent strains. Finally, the synergistic effect of <Mark2>icaA</Mark2> and <Mark2>icaD</Mark2> genes in the clinical <Mark2>S. aureus</Mark2> starines investigated here may be important to further understand slime layer formation and biofilm phenomena.</Pgraph></TextBlock> <TextBlock linked="yes" name="Notes"> <MainHeadline>Notes</MainHeadline><SubHeadline>Competing interests</SubHeadline><Pgraph>The authors declare that they have no competing interests.</Pgraph></TextBlock> <References linked="yes"> <Reference refNo="1"> <RefAuthor>Arciola CR</RefAuthor> <RefAuthor>Baldassarri L</RefAuthor> <RefAuthor>Montanaro L</RefAuthor> <RefTitle>Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheter-associated infections</RefTitle> <RefYear>2001</RefYear> <RefJournal>J Clin Microbiol</RefJournal> <RefPage>2151-6</RefPage> <RefTotal>Arciola CR, Baldassarri L, Montanaro L. 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DOI: 10.1007/978-3-540-75418-3_10</RefTotal> <RefLink>http://dx.doi.org/10.1007/978-3-540-75418-3_10</RefLink> </Reference> </References> <Media> <Tables> <NoOfTables>0</NoOfTables> </Tables> <Figures> <Figure format="png" height="577" width="368"> <MediaNo>1</MediaNo> <MediaID>1</MediaID> <Caption><Pgraph><Mark1>Figure 1: PCR results of</Mark1><Mark1><Mark2> ica</Mark2></Mark1><Mark1> gene for </Mark1><Mark1><Mark2>S. aureus</Mark2></Mark1></Pgraph></Caption> </Figure> <NoOfPictures>1</NoOfPictures> </Figures> <InlineFigures> <NoOfPictures>0</NoOfPictures> </InlineFigures> <Attachments> <NoOfAttachments>0</NoOfAttachments> </Attachments> </Media> </OrigData> </GmsArticle>