Detection of icaA gene for intracellular adhesion among clinical isolates of Staphylococcus aureus

 

Sri Vasavi Kadiyala¹, Dr. Gopinath P²

¹BDS  2nd year, Saveetha Dental College, Saveetha University, Chennai.

² Senior  Lecturer, Department of Microbiology, Saveetha Dental College, Saveetha University, Chennai.

 

ABSTRACT:

Staphylococcus aureus is a major hospital and community pathogen having the aptitude to cause a wide variety of infections in humans. 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 icaA 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. As the ability of this pathogen to adhere on to the various biotic and abiotic surfaces would eventually help in forming biofilm. In this study we studied the presence of icaA gene by PCR, by which 10% of our isolates were positive for it. This indicates the important role played by this gene in producing biofilm.

 

 

INTRODUCTION:

Staphylococcus aureus is one of the important bacterial pathogen causing a wide spectrum of infections ^[1]. Many studies have been conducted to explain the structures and pathogenic mechanisms by which S. aureus is able to cause serious infections ^[2]. The ability of S. aureus to produce biofilm enables this organism to withstand the host immune response and is considered to be the cause of many chronic or persistent infections, as the biofilm creation protects bacteria from phagocytosis and antimicrobial agents ^[3]. Another concern related to this pathogen is increasing resistance to oxacillin and many other antibiotics, but also circulation of multidrug resistant isolates within the hospital environment ^[4]. Staphylococcal pathogenesis is multifactorial, involving a combination of adherence and biofilm formation ^[5].

 

Complex aggregations of microorganisms can form irreversible attachments to the surfaces and formation of biofilm ^[6]. Biofilm producing bacteria are the source for persistent or chronic infections ^[4].

 

The significance of biofilm production for the virulence of S. aureus was supported by a number of clinical and animal studies ^[7]. 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.^[8] It was demonstrated that the ica-encoded genes are responsible for the biosynthesis of the PIA, which contains N-acetyl glucosamine as a main constituent and in the accumulation phase of biofilm formation, playing a crucial role in invasiveness of S. aureus ^[1]. Different studies have shown the decisive role of the ica gene as virulence factors in staphylococcal infections ^[2].

 

 

MATERIALS AND METHODS:

Bacterial isolates:

A total of 20 clinical isolates of S. aureus were collected from different clinical specimens of patients attending Saveetha Medical Collage and Hospital. They were processed for a battery of standard biochemical tests and confirmed. Isolates were preserved in semisolid trypticase soy medium and stored at 4ºC until further use.

 

Antibiotic susceptability test:

Antibiotic susceptibility testing was determined for these isolates to the following antibiotics such as penicillin, erythromycin, clindamycin, ciprofloxacin, tetracycline, cotrimoxazole and linezolid. These antibiotics were procured from Himedia, Mumbai. This was performed by Kirby-bauer disc diffusion method as per CLSI guidelines^[9].

 

Detection of icaA gene in Staphylococcus aureus:

Staphylococcus aureus isolates were detected for the presence of icaA gene by PCR analysis. Detection of the gene was carried out using primer as depicted in table 2. Bacterial DNA was extracted by boiling lysis method. 1 µL of DNA extract was used as template for PCR reaction. The reaction mixture contained 2mM of Mgcl₂ 0.2mM dNTP mix and 0.8µM of icaA gene with IU of Taq polymerase (New England Biolabs) in a 1x PCR buffered reaction. A positive control of S. aureus with icaA gene was also included in this study. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation at 95^oC for 1 min and 40 cycles for 30s,  51^oC for 60s and 72^o C for 30s, followed by a final extension of 6 min at 72^oC. PCR products were resolved in 1.5% agarose gel. A 100bp ladder was including in all the gel analysis ^[10].

 

Table 1: Primer detail of icaA gene

Primer	Primer sequence	Product size
icaA	5'-ACA GTC GCT ACG AAA AGA AA-3' 5'-GGA AAT GCC ATA ATG ACA AC-3'	103 bp

 

RESULTS:

Sample wise distribution of clinical isolates of S. aureus

 

Figure1: Sample wise distribution of S.aureus

Of 20 clinical isolates of S. aureus, 8/20 (40%) were obtained from pus, 6/20 (30%) were from wound, 4/20 (20%) and 2/20 (10%) were from blood and sputum respectively (Figure 1).

 

Antibiotic susceptibility pattern

We have observed a varied pattern of sensitivity among one S.aureus isolates. There was complete resistance observed for penicillin(100%), 9/20(45%)isolates were shown to the resistant to erythromycin,6/20(30%) were to cotrimoxazole,4/20(20%)were to linezolid followed by 3/20(15%) were resistant to ciprofloxacin and clindamycin respectively (Table 1) (Figure 2).

 

Table 2: Results of antibiotic susceptibility pattern of S.aureus

Antibiotics	Sensitive (%)	Intermediate (%)	Resistant (%)
Penicillin	0	0	20(100)
Erythromycn	14(70)	4(20)	2(10)
Clindamycin	15(75)	2(10)	3(15)
Ciprofloxacn	9(45)	8(40)	3(15)
Tetracyclin	14(70)	4(20)	2(10)
Cotrimoxazolee	10(50)	4(20)	6(30)
Linezolid	10(50)	6(30)	4(20)

 

 

 

 

 

 

 

 

Figure 2: Representative picture showing antibiotic sensitivity pattern of S. aureus

 

Result of icaA  gene in Staphylococcus aureus:

2/20 (10%) clinical isolate of S. aureus were found to possess icaA gene.

 

 

Zone of inhibition

 

 

Figure 3: Representative gel picture showing icaA gene

L1 – 100bp ladder, L3 and L4 – 103 bp icaA gene

 

 

DISCUSSION:

S. aureus is one of the most threatful organism causing infections in humans. It has been demonstrated that strains having an ability to form biofilm cause additionally “chronic polymer-associated” infection ^[7] , mostly seen as implant-associated infections. Biofilm support the adhesion and colonization of S. aureus on surfaces, frequently leading to persistent and difficult to eradicate infections ^[5].

 

Indeed, an increasing number of different S. aureus adhesion molecules is found ^[11]. The ica operon of S. aureus contain ica (ADBC) that allow Staphylococcus spp. to form slime layers and biofilm. The icaA is required to encode N-acetylglucosaminyltransferase, co expression of icaD can increase the capsular polysaccharide phenotypes ^[12], indicating a significant role of the icaD locus as a virulence factor in the pathogenesis of S. aureus isolated from catheters ^[13]. Infections associated with the use of invasive medical devices, e.g., catheters, are mainly due to S. aureus, particularly those strains which create an extracellular slime and parts of the biofilm, making clinical treatment extremely challenging.

 

The process of biofilm formation needs polysaccharide intercellular adhesion, which is synthesized by the enzymes encoded by the intercellular adhesion cluster (ica). In this study, the percentage of slime producing strains was %,while % of these had black colonies on CRA plate. Since the presence of adhesion molecules is required for the establishment 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 ^[14].

 

It can be concluded that infections caused by ica locus carrying S. aureus strains can lead to clinically difficult to treat conditions. The detection of the ica locus in clinical S. aureus isolates may improve the clinical decision for treatment and prevention options, and could support development of strategies to interact the bacterial capacity to colonise and invade in-dwelling medical devices. PCR detection of the ica operon may be an effective method to differentiate between virulent and non virulent strains. Finally, the synergistic effect of icaA genes in the clinical S. aureus strains investigated here may be important to further understand slime layer formation and biofilm phenomena. Study conducted by Park et al in 2008 from Korea have reported 76.9% of icaA gene positivity whereas, in our study we have observed only 10% of it.

 

CONCLUSION:

As different studies from different parts of the world have shown the decisive role of the ica gene as virulence factors in staphylococcal infections. Similarly we have also seen positivity of this gene in 2 isolates which indicates the cardinal role of this gene in forming biofilm in our isolates. Further this study has to be carried out with more number of samples to rule out the actual role of this gene. 

 

ACKNOWLEDGMENT:

We thank Dr. Kalyani, Professor and Head of the Department of Microbiology, Saveetha Medical College, Chennai for kindly providing the clinical isolates to carry out our research work fruitfully.

 

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Accepted on 20.07.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(9):1451-1453.