Detection of Biofilm Formation in Staphylococcus aureus. Does it have a role in Treatment of MRSA Infections?
Staphylococcus aureus is a causative agent of many types of diseases throughout the world. Staphylococcal infections are of particular concern because of the causative bacteria offering resistance to a wide range of commonly used antibiotics. The formation of biofilm is the hallmark characteristic of S. aureus infection. Biofilms constitute reservoir of pathogens and are associated with resistance to antimicrobial agents and chronic infections. In this study 262 clinical strains of S. aureus were screened by tissue culture plate method, tube method and congo red agar method for biofilm formation. Antimicrobial sensitivity testing of these strains was done by Kirby bauer disc diffusion method. Tissue culture plate method detected 38 (14.51%) isolates as strong biofilm producers, 132 (50.38%) as moderate biofilm producers and 92 (35.11%) strains as nonproducers of biofilm. The congo red agar method had a low sensitivity and specificity of 67.65 and 89.13%. The tube method correlated well with tissue culture plate method with a sensitivity and specificity of 99.40 and 95.78% but the interpretation are observer dependent. Biofilm production was higher in methicillin resistant strains as compared to the methicillin sensitive strains of S. aureus. Biofilm producers were found to be more resistant to almost all the groups of antibiotics.
February 22, 2011; Accepted: May 12, 2011;
Published: September 26, 2011
Staphylococcus aureus is a causative agent of many types of diseases
throughout the world. Staphylococcal infections are of particular concern
because of the causative bacteria offering resistance to a wide range of commonly
used antibiotics (Siddiqi et al., 2002). S.
aureus is consistently one of the top four causes of nosocomial infections
(Akhi et al., 2008). Patients hospitalized for
long periods of time are usually predisposed to infection by methicillin resistant
Staphylococcus aureus (Saeed and Ahmed, 2009).
At present approximately 40% of Staphylococcus aureus are resistant to
methicillin and the incidence of methicillin resistance increases year by year
(Akhi et al., 2008). MRSA poses increasingly
serious health care problem in many parts of the world. Several studies have
reported increased morbidity and mortality associated with MRSA compared to
methicillin sensitive Staphylococcus aureus infections (Dadgar
et al., 2006). Methicillin resistant strains of S.aureus are
more difficult to treat because multidrug resistance is more common in these
isolates as compared to the MSSA isolates. Vancomycin is considered as the treatment
of choice for MRSA cases but recently there are reports of emergence of vancomycin
resistance in S. aureus (Jasmine et al., 2007).
Staphylococcus aureus is an adaptable, pathogenic organism. In the presence
of environmental challenges, S. aureus can alter its genotype and/or
phenotype to adapt to its surroundings. An example of genotypic change is the
acquisition of the β-lactamase gene conferring penicillin resistance. The
formation of biofilm is an example of phenotypic change. Formation of a biofilm
is the hallmark characteristic of S. aureus infection which consists
of multiple layers of bacteria encased within an exopolysaccharide glycocalyx.
Presence of glycocalyx protects the enclosed bacteria from host defences and
impedes delivery of antibiotics (Stewart, 2002). Infact
biofilms can resist antibiotic concentration 10-10,000 fold higher than those
required to inhibit the growth of free floating bacteria (Jefferson
et al., 2005). Biofilm formation in S. aureus is regulated
by expression of Polysaccharide Intracellular Adhesion (PIA) which mediates
cell to cell adhesion and is the gene product of ica ABDC (Ammendolia
et al., 1999).
Infectious processes in which biofilms have been implicated include common
problems such as urinary tract infections, catheter infections, middle ear infections,
formation of dental plaque, gingivitis, coating contact lenses and less common
but more lethal processes such as infective endocarditis, cystic fibrosis and
infections of permanent indwelling devices such as joint prosthesis and heart
valves (Lewis, 2001; Parsek and
The present study was undertaken to detect the prevalence of biofilm production in S. aureus in a tertiary care hospital in North India and to evaluate three different methods, viz., Tissue Culture Plate method (TCP), Tube Method (TM) and Congo Red Agar (CRA) method for detection of biofilms and to see its relation with antimicrobial resistance.
MATERIALS AND METHODS
A total of 262 non-repetitive, clinical strains of S. aureus were isolated
from various clinical samples of the indoor and outdoor patients of Jawaharlal
Nehru Medical College and Hospital, Aligarh, India during one year period from
August 2006 to August 2007. Isolates were identified by standard microbiological
techniques including gram stain, catalase test, coagulase test, phosphatase
test and DNAase test (Baird, 2006). All the isolates
were classified into hospital and community acquired S. aureus. S.
aureus was considered as community acquired when it was isolated from the
out patient setting or by a culture positive for S. aureus within 48
h after admission to the hospital with no history of permanent indwelling catheters
or medical devices that pass through the skin into the body, any medical history
of MRSA infection or colonization, any history in the past year of hospitalization,
admission to a nursing home or skilled nursing facility, dialysis unit or for
surgical intervention. Otherwise the patient was considered to have hospital
Oxacillin disc diffusion test: All the isolates were subjected to oxacillin disc diffusion test using oxacillin 1 μg disc. A 0.5 McFarland turbidity standard suspension of the isolate was made and lawn culture was done on Mueller-Hinton Agar (MHA) plates containing 4% NaCl. Plates were incubated at 37°C for 18 h and zone diameters were measured. An inhibition zone diameter of ≤10 mm was reported as methicillin resistant and ≥13 mm was taken as methicillin sensitive.
PCR amplification for mec A and fem B genes: Multiplex PCR (Geha
et al., 1994) was carried out on all the S. aureus strains
found methicillin resistant on MIC determination. All the MRSA strains were
screened for the mec A and femB genes using the following oligonucleotides
sequence.mec A1-5 GTA GAA ATG ACT GAA CGT CCG ATA A-3, mec
A2-5 CCA ATT CCA CAT TGT TTC CGT CTA A-3, fem B1-5
TTA CAG AGT TAA CTG TTA CC-3, fem B2-5 ATA CAA ATC CAG CAC
GCT CT-3. A 50 μL PCR reaction mixture consisted of 45 μL of
mastermix containing PCR buffer (1X), dNTP mix (0.2 mM of each), primer (0.5
μM), Taq DNA polymerase (0.25 U) and MgCl2 (1.5 mM) with 5
μL of template DNA. Cycling parameters were set to- hot start 94°C
for 4 min followed by 35 cycles of melting at 94°C for 45 sec, annealing
at 50°C for 45 sec and extension at 72°C for 1 min. Analysis of amplified
products was done by gel electrophoresis. Amplicons of 310 bp were consistent
with mec A and of 651 bp with fem B gene amplification.
Antimicrobial sensitivity testing: Antimicrobial sensitivity testing was done by Kirby bauer disc diffusion method for the following antimicrobial agents: amikacin 30 μg, ciprofloxacin 5 μg, clindamycin 2 μg, cotrimoxazole 25 μg, erythromycin 15 μg, gatifloxacin 5 μg, gentamycin 10 μg, levofloxacin 5 μg, linezolid 30 μg, ofloxacin 5 μg, sparfloxacin 5 μg, tobramycin 10 μg, vancomycin 30 μg.
Detection of biofilm formation was done by the following methods:
Statistical analysis: Statistical analysis was done using Microsoft
A total of 262 clinical strains of S. aureus were isolated from various clinical samples, of which 217 (82.83%) samples were hospital acquired and 45 (17.17%) were community acquired. Out of the total of 262 S. aureus isolates tested for methicillin susceptibility 89 (33.97%) were found to be resistant to methicillin (MRSA) whereas 173 (66.03%) were sensitive (MSSA) by oxacillin disc diffusion method. However, 85 (32.44%) strains had both mec A (310 bp) and fem B (651 bp) and were confirmed to be methicillin resistant. MRSA was found to be more prevalent in hospital acquired isolates 72 (33.17%) of S. aureus. Among the OPD patients rate of isolation of MRSA was 28.89% (Table 1).
Biofilm production by S. aureus: Out of the 262 isolates tested for biofilm formation by CRA method, 182 isolates produced black colonies. However, only 125 (47.71%) colonies were black in colour with dry crystalline consistency which is indicative of biofilm formation. The 57 (21.76%) isolates were black in colour but were not dry and crystalline and were indeterminate for biofilm formation. These isolates were also taken as negative for biofilm formation. The 80 (30.53%) isolates produced pink colonies which were taken as negative for biofilm formation (Table 2, Fig. 1a).
Out of 262 isolates tested for biofilm by Tube Method, 35 (13.36%) were strongly positive. However, maximum number of isolates 132 (50.38%) were moderately positive and 95 (36.26%) did not show any biofilm formation (Table 3, Fig. 1b).
|| Isolation rate of MRSA in relation to hospital and community
||Biofilm formations by S. aureus (Congo red agar method)
(n = 262)
||Biofilm formation by S. aureus (Tube method)
||(a) Biofilm producer and nonproducer on Congo-red agar, (b)
Biofilm production by tube method with positive and negative control and
(c) Tissue culture plate method for detection of biofilm production
By tissue culture plate method, 38 (14.51%) isolates were strongly positive for biofilm production, 132 (50.38%) were moderate biofilm producers whereas 92 (35.11%) were negative for biofilm formation (Table 4, Fig. 1c).
When the results of CRA and Tube method for biofilm formation were compared with those of tissue culture plate method, it was found that the specificity and sensitivity of CRA was 89.13% and 67.65% and for Tube method, the specificity and sensitivity were 95.78 and 99.40% (Table 5).
||Statistical evaluation of TM and CRA for detection of biofilm
formation in S. auerus
|*Comparative evaluation of Tube method and CRA method with
TCP method for detection of biofilm formation in S. aureus (n = 262)
Antimicrobial susceptibility profile in relation to biofilm production: It was found that biofilm producing strains were more resistant when compared to the biofilm non producers (Fig. 2). All the strong biofilm producers thirtyeight (by tissue culture plate method) were found to be methicllin resistant. Out of the remaining 47 MRSA strains 40 were moderate biofilm producers and just 7 (8.23%) strains did not produced any biofilm. Amongst the 177 MSSA strains 92 strains (51.98%) were found to be moderate biofilm producers and none was strong producer of biofilm. The resistance pattern of biofilm producing strains when compared to biofilm non producers was for amikacin 73.53/55.43%, chloramphenicol 54.94/34.78%, ciprofloxacin 83.53/76.09%, clindamycin 87.79/78.26%, cotrimoxazole 93.60/79.35%, erythromycin 65.29/53.26%, gatifloxacin 48.23/40.22%, gentamycin 70.00/67.39%, levofloxacin 12.35/6.42%, ofloxacin 24.71/21.74%, sparfloxacin 43.53/33.69%. However, all the strains were sensitive to linezolid and vancomycin.
Bioilm formation is an important characteristic of all staphylococcal species,
associated with the infection of biomedical devices (Christensen
et al., 1985; Kloos and Bannerman, 1994;
OGara and Humphreys, 2001). Polysaccharide Intercellular
Adhesion (PIA) plays an important role in the pathogenesis as it mediates the
contact of bacterial cells with each other, resulting in the accumulation of
a multilayered biofilm (Chaudhary et al., 2009).
Biofilms constitute reservoir of pathogens and are associated with resistance
to antimicrobial agents and chronic infections.
In present study, a total of 262 strains of S. aureus were isolated,
out of which 85 (32.44%) were methicillin resistant. More than 2/3rd of the
isolates were from patients having hospital acquired infections (around 80%).
Community acquired isolates accounted for just 17% of the isolates of S.
aureus (Table 1). Among the hospital acquired isolates
33.17% were found to be methicillin resistant whereas among the community acquired
isolates around 29% isolates were resistant to methicillin. Methicillin resistant
S. aureus is a known cause of nosocomial infections, however, recently
there has been emergence of community acquired infections associated with MRSA
(Eguia and Chambers, 2003; Oberoi
and Verghese, 2006). It was also noted that methicillin resistant strains
of S.aureus were more prone to biofilm formation as compared to themethicillin
sensitive strains of S. aureus. Methicillin susceptibility of S. aureus
has been shown to influence the biofilm formation (ONeill
et al., 2007).
|| Antimicrobial resistance of biofilm producers and non-producers
On antimicrobial sensitivity testing by Kirby bauer disc diffusion method it
was noted that biofilm forming strains showed higher resistance to almost all
the groups of antibiotics as compared to the biofilm non-producers (Fig.
2). The increased resistance of biofilm producing strains to antibiotics
may be because the biofilm bacteria exhibit a slow rate of metabolism and divide
infrequently resulting in decreased sensitivity to antibiotics targeted at cell
wall synthesis (Monroe, 2007). However, even antibiotics
targeted at cellular functions such as protein and DNA synthesis which should
affect cells at a quiescent state are ineffective against biofilms (Lewis,
All the 262 isolates of S. aureus were subjected to three in-vitro
screening procedures for their potential to form biofilm. By the tissue culture
plate method 170 (64.89%) isolates were found to be biofilm producers and 92
(35.11%) strains did not produced biofilm (Table 4). However,
other studies have reported a slightly less number of biofilm production by
staphylococcal species (Mathur et al., 2006;
Bose et al., 2009). By tube method, biofilm formation
was observed in 167 (63.74%) isolates whereas 95 (36.26%) strains did not show
any biofilm formation (Table 3). In our study positivity for
biofilm formation by CRA method was 125 (47.79%) (Table 2)
which is much higher than reported by Mathur et al.
(2006) and Bose et al. (2009).
Highly accurate methods like PCR for detection of ica gene are available
to check the capability of S. aureus strains to produce biofilm but these
are beyond the scope of most of the microbiology laboratories in the developing
countries like India. Among the phenotypic methods Tissue culture plate method
has been reported as gold standard for biofilm formation (Mathur
et al., 2006). We compared the results of tube method and CRA method
to the tissue culture method for biofilm formation. It was noted that both the
sensitivity and specificity of tube method (95.78/99.40%) were higher than those
for CRA method (89.13/67.65%).
CRA method is easy to perform and interpret but due to its low specificity
and sensitivity we do not recommend it for detection of biofilm formation. Tube
method has high sensitivity and sensitivity and the results of the tube test
correlate well with the TCP method but the interpretation is observer dependent
and there are chances of subjective errors. When S. aureus assumes the
biofilm phenotype, these infections are often extremely difficult to treat.
The infection may fail to respond to antibiotic therapy or it may initially
respond only to relapse weeks or months later. In such cases, invasive treatments,
such as surgical removal and replacement of the infected tissue or device, may
be required. So for proper treatment of S. aureus infection screening
for biofilm production is necessary. However, this might not be feasible in
every case, so we recommend that in all cases of methicillin resistant S.
aureus infections and in patients with hospital acquired staphylococcal
infection screening for biofilm should be done routinely by tissue culture plate
method as this is a cheap method with no subjective errors and requires less
Treatment of MRSA is one of the most challenging task for the clinicians and the microbiologists. With the emergence of vancomycin resistance in Staphylococcus aureus role of antimicrobials is becoming limited. Hospital acquired strains of MRSA should be routinely screened for biofilm formation using the tissue culture plate method and treated accordingly.
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