Detection of PVL gene for the presence of Leukocidin among Clinical Isolates of Staphylococcus aureus from Tertiary Care Hospital

 

Gowtham R¹, Dr. P. Gopinath²*

¹BDS, Saveetha Dental College, Chennai.

²Senior Lecturer, Department of Microbiology, Saveetha Dental College, Chennai, Tamil Nadu, India

 

ABSTRACT:

PVL and γ -haemolysin are considered to be members of a toxin family known as synergohymenotropic toxins, as they act on cell membranes by the synergy of two proteins that form a pore. Only 2% of S. aureus isolates produce PVL, while γ -haemolysin is produced by more than 99% of S. aureus isolates. PVL is the most leukocytolytic toxin in the family, however it does not exhibits no haemolytic activity on human erythrocytes. A sum of 20 clinical isolates of S. aureus were subjected to antibiotic sensitivity pattern followed by the detection of pvl gene by PCR. We have observed increased resistance to most of the routinely used antibiotics and 10% of our isolates found to have pvl gene. As this gene is directly associated with skin and soft tissue infections by S. aureus, our two isolates may even cause such infections, although none of these strains were not obtained from cutaneous lesions.

 

 

 

INTRODUCTION:

Staphylococcus aureus is an important human pathogen that causes a wide range of diseases from mild superficial skin infection to life-threatening bacteremia and infective endocarditis, as well as toxin-mediated conditions such as toxic shock syndrome.^[1] They produce more than 30 different extracellular products.^[2] Nearly all strains exhibit a group of enzymes and cytotoxins that includes haemolysins (α, β, γ and δ), nucleases, proteases, lipases, hyaluronidase and collagenase. Some strains produce one or more additional exoproteins, which include toxic shock syndrome toxin-1 (TSST-1), the staphylococcal enterotoxins (SEA-E, G-I), the exfoliative toxins (ETA and ETB) and Panton–Valentine leukocidin (PVL).^[3]

 

PVL and γ -haemolysin are considered to be members of a toxin family known as synergohymenotropic toxins, as they act on cell membranes by the synergy of two proteins that form a pore.

 

 

Only 2% of S. aureus isolates produce PVL, while γ -haemolysin is produced by more than  99% of S. aureus  isolates.^[4] Pvl is the most leukocytolytic toxin in the family, however it does not exhibits no haemolytic activity on human erythrocytes.^[5] It also dermo-necrotic, as observed after intradermal injection of rabbit skin.^[6] At sub-lytic concentrations, PVL has been demonstrated to induce granule secretion and release of leukotriene B4 and interleukin-8 from human polymorphonuclear leukocytes.^[6] Based on this background, we have undertaken this study to detect the presence of pvl gene among S. aureus by PCR.

 

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 4°C 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^[7]

 

Detection of pvl gene in Staphylococcus aureus:

Staphylococcus aureus isolates were detected for the presence of pvlgene by PCR analysis. Detection of the gene was carried out using primer as depicted in table 1. 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 pvlgene with IU of Taq polymerase (New England Biolabs) in a 1x PCR buffered reaction. A positive control of S. aureus with pvl gene was also included in this study. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation at 96^oC for 2 min and 30 cycles for 30s, 52^oC for 60s and 73^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.^[8]

 

Table 1: Primer detail of pvl gene

Primer	Primer sequence	Product size
pvl	ATC ATT AGG TAA AAT GTC TGG ACA TGA TCC A GCA TCA AST GTA TTG GAT AGC AAA AGC	433 bp

 

RESULTS:

Sample wise distribution of clinical isolates 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).

 

Figure 1: Pie chart showing the sample wise distribution of S. aureus

 

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 2).

 

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

Antibiotics	Sensitive (%)	Intermediate (%)	Resistant (%)
Penicillin	0	0	20(100)
Erythromycin	14(70)	4(20)	2(10)
Clindamycin	15(75)	2(10)	3(15)
Ciprofloxacin	9(45)	8(40)	3(15)
Tetracyclin	14(70)	4(20)	2(10)
Cotrimoxazole	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 pvl  gene in Staphylococcus aureus:

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

 

Figure 3: Representative gel picture showing pvl gene

 

DISCUSSION:

In the present study, we have detected pvl gene in 2/20 (10%) of our S. aureus isolates.  Similar kind of study conducted by Johnsson and colleagues in 2004 reported PVL gene was detected in one out of 65 S. aureus isolates collected prospectively from septicaemic patients.^[9] This finding was in agreement with our study and other previous studies showing prevalences of 0–2% in positive blood cultures.^[10] In addition, none of the S. aureus isolates causing infective endocarditis was PVL-positive.^[11] Thus, PVL does not seem to represent an important virulence factor in invasive bloodstream infections. Our isolates were not collected from skin and soft tissue related infection, even though, two strains were showed positive for this gene. In cutaneous infections, PVL has been associated more frequently with direct invasion and tissue destruction (e.g., necrotising primary skin infections such as furunculosis) than with secondary infections after skin injury.^[12] PVLpositive S. aureus is isolated rarely from cases of folliculitis or impetigo.^[13]

 

CONCLUSION:

In our study, we have seen only 10% of them were found to harbor pvl gene. As this gene is directly associated with skin and soft tissue infections by S. aureus, our two isolates may even cause such infections, although none of these strains were not obtained from cutaneous lesions. In order to prove its virulence, it is necessary to include more samples especially from cutaneous lesions.

 

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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13.   Pre´vost G, Couppie´ P, Pre´vost P et al. Epidemiological data on Staphylococcus aureus strains producing synergohymenotropic toxins. J Med Microbiol 1995: 42, 237–245.

 

 

 

 

 

Accepted on 17.10.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(1): 172-174.