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Int. J. Pharm. Med. & Bio. Sc. 2014 Sarita Yadav et al., 2014

A STUDY OF BIOFILM PRODUCTION IN

STAPHYLOCCUS AUREUS

Sarita Yadav1*, Madhu Sharma2, Aparna Yadav2 and Uma Chaudhary2

Research Paper

Aims and objectives: (1) To detect biofilm formation in clinical isolates of Staphylococcus aureus.(2) To know the antimicrobial resistance pattern of the isolates and correlation with biofilmproduction. Methodology: The present study was done to detect biofilm formation in 50 clinicalisolates of Staphylococcus aureus from blood and indwelling devices. For detection of biofilmformation, the clinical isolates were screened by Modified Tissue Culture Plate method (MTCP)and Tube Method (TM).Antibiotic susceptibility of the isolates was determined by using Kirby-Bauer disc diffusion method. Results: Out of 50 isolates of Staphylococcus aureus, 46% werebiofilm positive by MTCP and 38% by TM. The biofilm producers were multi drug resistant ascompared to the non producers. Interpretation: The effective control for staphylococcal infectionswill require a concerted effort to develop therapeutic agents that target the biofilm phenotype.

Keywords: S. aureus, Biofilms, Multidrug resistance, Antibiotics

*Corresponding Author: Sarita Yadav � yadav78sarita@yahoo.com

INTRODUCTION

Biofilms are complex communities of single or

multiple species of micro-organisms that develop

on biotic and abiotic surfaces. Since biofilms

contaminate catheters, ventilators and medical

implants, they act as a source of diseases for

humans. Staphylococcus aureus and

Staphylococcus epidermidis are the predominant

species forming biofilms on polymeric surfaces

(Kloos and Bannerman, 1994). The genetic and

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Int. J. Pharm. Med. & Bio. Sc. 2014

1 Department of Microbiology BPS, GMC for Women, Khanpur Kalan, Sonepat, Haryana.

2 Department of Microbiology, Pt. B.D. Sharma PGIMS, Rohtak, Haryana, India.

molecular basis of biofilm formation in

staphylococci is multifaceted. The ability to form

a biofilm affords at least two properties: the

adherence of cells to a surface and accumulation

to form multilayered cell clusters. A trademark is

the production of slime substance PIA, a

polysaccharide composed of β-1,6-linked N-

acetyl glucosamines with partly deacetylated

residues in which the cells are embedded and

protected against the host’s immune defence and

antibiotic treatment (Gotz, 2002).

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Int. J. Pharm. Med. & Bio. Sc. 2014 Sarita Yadav et al., 2014

Methicillin Resistant Staphylococcus aureus

(MRSA) isolates in hospitals/communities have

been recognized as the major challenges as they

are becoming Multi Drug Resistant (MDR) (Mehta

et al., 1998). Therefore, keeping this in view, the

present study was designed to detect the

production of biofilm by Staphylococcus aureus

and its correlation with antibiotic resistance.

METHODOLOGY

A total of 50 non repetitive, clinical isolates of

Staphylococcus aureus isolated from blood and

infected indwelling devices received in

Microbiology Department, Pt.BDS, PGIMS,

Rohtak, Haryana were investigated. Organisms

were identified by standard microbiological

techniques (Collee et al., 1996). Antibiotic

susceptibility test was performed by Kirby Bauer

disc diffusion method (CLSI). Biofilm production

was detected by using two methods: (1) Modified

Tissue Culture Plate (MTCP) method (Mathur et

al., 2006) by using crystal violet binding assay in

a 96-well flat bottomed tissue culture plate and

quantitated spectrophotometrically using ELISA

reader at 600 nm; and (2) Tube Method (TM)

(Christensen et al., 1982) in which biofilm

formation was taken as positive when a visible

film lined the wall and bottom of the tube.

RESULTS

In our study, by MTCP method we could detect

biofilm formation in 23 isolates (46.0%). By tube

method, 19 isolates (38%) were positive for

biofilm formation.

Out of 50 isolates of Staphylococcus aureus,

33 (66.0%) were MRSA, of which 20(60.6%) were

positive for biofilm production. Also MDR was

more frequent in biofilm producers in comparison

to non-biofilm producers.

DISCUSSION

Infections by Staphylococcus aureus are a major

problem in hospital settings, especially among

patients with indwelling devices. Most serious

infections such as endocarditis, osteomyelitis and

catheter-related infections are caused by biofilm

producing strains. Such infections are difficult to

manage and costlier to treat (Gara and

Humphreys, 2001). Over the last few years,

several studies have been performed to

demonstrate the biofilm formation by

staphylococci ( Ammendolia et al., 1999; Ruzicka

et al., 2004).

We tested 50 clinical isolates of

Staphylococcus aureus by two in-vitro screening

procedures for their ability to form biofilms. In the

MTCP method, biofilm production was seen in

46.0% which is less than that by other workers

who demonstrated biofilm formation in 53.9% and

57.1% of isolates respectively (Mathur et al., 2006;

Ammendolia et al., 1999). By tube method, we

could detect biofilm formation in 38.0% isolates,

while other studies have demonstrated 41.4% and

53.7% positivity rate respectively (Mathur et al.,

2006; Ruzicka et al., 2004).

In the present study, biofilm was more

common in MRSA isolates. The biofilm producers

exhibited significantly higher resistance to all the

antibiotics which is in concordance with other

studies (Smith and Hunter, 2008; Campoccia et

al., 2006). We did not observe resistance to

linezolid. Linezolid have been evaluated in many

in-vitro biofilm models and it has been

demonstrated that there is suppression of

bacterial growth in a biofilm, but it does not

eradicate bacterial colonization.

Biofilm is one of the known virulence factors

of staphylococci. So a greater understanding of

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Int. J. Pharm. Med. & Bio. Sc. 2014 Sarita Yadav et al., 2014

the nature of intracellular bacterial communities

in infections, their early detection and

management will aid in the development of new

and more effective treatments.

CONCLUSION

Microbial biofilms pose a public health problem

especially in persons with indwelling devices.

Therefore, an effectual control will need an

intensive effort to build up newer therapeutic

agents that aim to prevent the formation of biofilms

or encourage the biofilm detachment.

REFERENCES

1. Ammendolia M G et al. (1999), ”Slime

production and expression of the slime-

associated antigen by staphylococcal

clinical isolates”, J Clin Microbiol, Vol. 37,

pp. 3235-3238.

2. Campoccia D et al. (2006), ’’The

significance of infection related to

orthopaedic devices and issues of antibiotic

resistance”, Biomaterial, Vol. 27, No. 11, pp.

2331-2339.

3. Christensen G D et al. (1982), ”Adherence

of slime producing strains of Staphylococcus

epidermidis to smooth surfaces”, Infect

Immun, Vol. 37, pp. 318-326.

4. CLSI (Clinical and Laboratory Standards

Institute) (2007), Performance standards for

antimicrobial disc susceptibility tests, 9th

Edn. Approved Standard M2-A9.Wayne, PA:

document M100-S17.

5. Collee J G, Duguid J P, Fraser A G, Marmion

B P and Simmons A (1996), in Collee J G,

Fraser A G, Marmion B P and Simmons AC

(Eds.), Mackie and McCartney Practical

Medical Microbiology, Churchill Livingstone,

New York, pp. 53-94.

6. Gara O J and Humphreys H (2001),

”Staphylococcus epidermidis biofilms:

importance and implications”, J Med

Microbiol, Vol. 50, pp. 582-587.

7. Gotz F (2002), ” Staphylococcus and

biofilms”, Mol Microbiol, Vol. 43, No. 6, pp.

1367-1378.

8. Kloos W E and Bannerman T L (1994),

”Update on clinical significance of

coagulase negative staphylococci”, Clin

Microbiol Rev, Vol. 7, pp. 117-140.

9. Mathur T et al. (2006), ”Detection of biofilm

formation among the clinical isolates of

staphylococci: An evaluation of three

different screening methods”, Indian J Med

Microbiol, Vol. 24, No. 1, pp. 25-29.

10. Mehta A Pet al. (1998), ”Control of methicillin

resistant Staphylococcus aureus in a tertiary

care centre-A five year study”, J Med

Microbiol, Vol. 16, pp. 31-34.

11. Ruzicka F et al. (2004), ”Biofilm detection

and clinical significance of Staphylococcus

epidermidis isolates”, Folia Microbiologica,

Vol. 49, No. 5, pp. 596-600.

12. Smith K and Hunter L S (2008), ”Efficacy of

common hospital biocides with biofilms of

MDR clinical isolates”, J Med Microbiol,

Vol. 57, pp. 966-973.