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 Table of Contents  
Year : 2022  |  Volume : 14  |  Issue : 1  |  Page : 43-47

Phenotypic Detection of Biofilm Formation in Clinical Isolates of Methicillin-Resistant Staphylococcus aureus

1 Department of Microbiology, Santosh Medical College and Hospital, Santosh Deemed to be University, Ghaziabad, Uttar Pradesh, India
2 Department of Biochemistry, Santosh Medical College and Hospital, Santosh Deemed to be University, Ghaziabad, Uttar Pradesh, India

Date of Submission05-Dec-2021
Date of Decision25-Jan-2022
Date of Acceptance26-Jan-2022
Date of Web Publication01-Mar-2022

Correspondence Address:
Ashutosh Rawat
Department of Microbiology, Santosh Medical College and Hospital, Santosh Deemed to be University, Ghaziabad - 201 009, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajprhc.ajprhc_15_21

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Background: Staphylococcus aureus is one of the common pathogens which causes mild-to-severe diseases. Moreover, its ability to produce biofilm along with drug resistance makes it more notorious and difficult to treat; therefore, early biofilm detection can help in better management of diseases caused by methicillin-resistant S. aureus (MRSA). Materials and Methods: All clinically isolated S. aureus were screened for their antibiotic susceptibility pattern followed by MRSA status by cefoxitin disk-diffusion method. Evaluation of biofilm-producing nature of biofilm was accessed by tissue culture plate (TCP), tube adhesion (TA), and Congo red agar (CRA) methods. Results: Of all S. aureus isolates, 57% had MRSA status. TCP method showed that 68% of MRSA were biofilm producers. TA method showed 55% and CRA method showed 46% biofilm production. Conclusion: Early detection of MRSA and biofilm could be very beneficial to control S. aureus infection. Of all three biofilm detection methods, TCP method was effective in detecting biofilm, followed by TA and CRA methods.

Keywords: Biofilm, Congo red agar culture method, methicillin-resistant staphylococcus aureus, Staphylococcus aureus, tissue culture plate method, tube adhesion method

How to cite this article:
Malik N, Bisht D, Aggarwal J, Rawat A. Phenotypic Detection of Biofilm Formation in Clinical Isolates of Methicillin-Resistant Staphylococcus aureus. Asian J Pharm Res Health Care 2022;14:43-7

How to cite this URL:
Malik N, Bisht D, Aggarwal J, Rawat A. Phenotypic Detection of Biofilm Formation in Clinical Isolates of Methicillin-Resistant Staphylococcus aureus. Asian J Pharm Res Health Care [serial online] 2022 [cited 2022 May 25];14:43-7. Available from: http://www.ajprhc.com/text.asp?2022/14/1/43/338789

  Introduction Top

Staphylococcus aureus is a Gram-positive coccus, which is a part of normal flora of the skin, gastrointestinal tract, and upper respiratory tract, especially nares. S. aureus causes a wide range of diseases from localized suppurative skin infection to life-threatening systemic infections, and it is one of the main bacterial agents of hospital-acquired infections. S. aureus is frequently resistant to most of the commonly used beta-lactam antibiotics.[1],[2] Methicillin-resistant S. aureus (MRSA) is an established multidrug-resistant bacterium, prevalence ranging from 4.6% to 54.4%.[3] One of the probable reasons for such resistance is that S. aureus is capable of forming a biofilm. A loose-bound, water-soluble film (biofilm) consisting of monosaccharides, proteins, and small peptides is produced by most staphylococci in varying amounts. This extracellular substance binds the bacteria to tissues and foreign bodies such as catheters, grafts, prosthetic valves and joints, and shunts and is particularly important for the survival of S. aureus as it protects bacteria from environment, antimicrobials, and host response.[4],[5] Detection of biofilm-producing S. aureus might help in effective disease prevention and better management.[6],[7] Therefore, the present study aims to detect biofilm formation in MRSA by different phenotypic methods.

  Materials and Methods Top

The present study was carried out in the Department of Microbiology, Santosh Medical College, UP. Approval of the study was obtained from the IEC (Institutional Ethics Committee-SU/R/2021/1844) through the proper channel. All pus specimens from in and outpatient departments of our tertiary care hospital were included for the present study. All samples were processed as per the standard microbiological procedure for the isolation and identification of S. aureus.[8]

Antibiotic susceptibility testing

It was performed on Mueller–Hinton agar using the standard Kirby–Bauer method and different groups of antibiotics were tested against S. aureus. Zones were interpreted as per the Clinical and Laboratory Standards Institute guidelines.[9]

Methicillin-resistant staphylococcus aureus detection by cefoxitin disk (30 μg) diffusion method

S. aureus was considered to be a MRSA if the cefoxitin zone of inhibition was ≤21 mm.[10]

Phenotypic detection of biofilm production in methicillin-resistant staphylococcus aureus

Tissue culture plate method

The isolates were placed in brain–heart infusion broth and incubated for 24 h at 37°C. Biofilm production was detected in a microtiter plate. First, 200 μl of the brain–heart infusion broth was added to each well. The wells were then filled with 20 μl of each sample to obtain 105cfu/ml as a final concentration and incubated at 37°C for 24 h. Well contents were discarded and removed by tapping. Then, 200 μl phosphate-buffered saline was used to wash each well four times. Then, 100 μl of 0.1% crystal violet was added to each well to stain it, and it was left for 15 min as shown in [Figure 1]. The wells were allowed to dry, then reading was taken using an ELISA plate reader at 570 nm. The reading values are interpreted as[11] Optical density of growth control (ODc):
Figure 1: Tissue culture plate method showing biofilm production by Staphylococcus aureus

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  • No biofilm producer = Optical density (OD) ≤ ODc
  • Weak biofilm producer = ODc < OD ≤ 2× ODc
  • Moderate biofilm producer = 2× ODc < OD ≤ 4× ODc
  • Strong biofilm producer = 4× ODc < OD.

Tube adherence method

The isolated organisms were inoculated in 5 mL trypticase-soy broth in test tubes and further incubated overnight at 37°C along with the control organism, after incubation; the tubes were decanted, dried, and stained with 0.1% CV. After that, tubes were gently washed and placed upside down for drying. Visible CV-stained area of the wall and bottom of the tube was considered as positive for biofilm production. The results were accessed visually as weak, moderate, and strong biofilm producers or no biofilm producers.[12]

Congo red Agar culture method

The MRSA was cultured on Congo red agar (CRA) plates and incubated at 37°C in aerobic conditions for 24 h. The formation of black colonies on CRA plates was considered as biofilm production, whereas nonproducing strains appeared as smooth, pinkish-red colonies on CRA as shown in [Figure 2].[13]
Figure 2: Congo red agar showing biofilm production by Staphylococcus aureus

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  Results Top

A total of 222 S. aureus were isolated from pus specimens, of these isolates, 127 were MRSA (57%). All MRSA (127) isolates were further tested for their biofilm production by three different phenotypic methods. Tissue culture plate (TCP) method demonstrated that 68% of MRSA were biofilm producers. Among them, majority 72% were strong film producers, 22% of isolates were moderate, and 6% were weak biofilm producers. Tube adhesion (TA) method showed that 55% MRSA were positive for biofilm production. Among them, 64% were strong film producers, 21% were moderate, and 14% were weak biofilm producers. CRA method detected 46% MRSA isolates as biofilm producers. Among them, 48% were strong, 38% were moderate, and 14% were weak biofilm producers, as described in [Table 1].
Table 1: Biofilm production in methicillin-resistant Staphylococcus aureus (127)

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The antibiotic susceptibility testing pattern of biofilm and nonbiofilm-producing MRSA isolates is summarized in [Table 2] and [Table 3]. Linezolid and teicoplanin were the most effective antibiotics against biofilm-producing MRSA, followed by clindamycin, gentamicin, chloramphenicol, and tetracycline. On the other hand, linezolid and teicoplanin were the most effective antibiotics against nonbiofilm-producing MRSA, followed by gentamicin, tetracycline, ciprofloxacin, and clindamycin.
Table 2: Antibiotic susceptibility testing pattern of biofilm-producing methicillin-resistant Staphylococcus aureus

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Table 3: Antibiotic susceptibility testing pattern of nonbiofilm-producing methicillin-resistant Staphylococcus aureus

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  Discussion Top

MRSA is one of the common causes of nosocomial infection that causes high mortality and morbidity in patients; moreover, MRSA limits the therapeutic option for the treatment.[14] The prevalence of MRSA varies from place to place and hospital to hospital as there are so many factors which contribute in the occurrence of MRSA. The present study showed 57% occurrence of MRSA. Khadri and Alzohairy reported a 55% prevalence of MRSA which was close to our finding.[15] The MRSA prevalence of 28.8% and 29.1% has been reported by Uma Devi S.[16] and Pai et al.,[17] respectively, which were lower than the current study, whereas research done by Savitha et al.[18] and Verma et al.[19] revealed 62% and 81% prevalence of MRSA, respectively, which were on the higher side compared to present research. S. aureus with biofilm-producing capacities is capable of causing many recalcitrant infections, and they are difficult to eradicate and treat. They show antibiotic resistance by several mechanisms such as slow bacterial growth rate, acquired resistant gene from other bacteria, and slow/less diffusion of antibiotics into the biofilm matrix.[20] There are various methods and techniques available to detect the biofilm formation by phenotypically or genotypically. In the current study, we evaluate biofilm detection by three different phenotypic methods. TCP method considers as a best phenotypic method for biofilm detection.[21] We also found that TCP method was able to detect more biofilm producers MRSA isolates (68%) compared to TA (55%) and CRA (46%) methods. Prevalence differences (<50 to >70%) in the biofilm-producing S. aureus have been reported by several studies which were concordant with our results.[22] Hassan et al.[23] demonstrated 65% biofilm-producing MRSA using the TCP method which was close to our study, whereas Mathur et al.[24] reported 54% biofilm production which was comparatively less than the present study. Abdel Halim et al.[25] reported 43% biofilm detection by TA-method which was less than our study (55%). On the other hand, Hassan et al. revealed 58% which was close to our results.[23] Tahaei et al.[26] from Hungary documented that 44% of MRSA isolates were biofilm producers when they were screened by CRA-method which was concordant with the present study. The present study demonstrated that biofilm-producing MRSA isolates were more resistant to antibiotics which are commonly used for the treatment than the nonbiofilm-producing isolates. Abdel Halim et al.[25] and and Singh et al.[27] also concluded that biofilm producers were more resistant isolates than nonproducers. In contrast to the present study Tahaei et al.[26] documented that they could not find any stabilized relation between drug-resistant pattern of biofilm-producing and nonproducing MRSA isolates.

  Conclusion Top

Biofilm production by S. aureus is an important virulence factor in producing various infections. Treatment of infections caused by MRSA-producing biofilm is a therapeutic challenge as embedment of MRSA in slime layers gives bacteria an increased tolerance to antibiotics; therefore, the understanding of S. aureus forming a biofilm is necessary in order to develop new strategies to inhibit the biofilm formation. Current research is bringing additional important information regarding the biofilm-producing status of MRSA strains and their association with antibiotic resistances in a particular area. TCP method was the best method to screen biofilm production in MRSA as it discriminates well between strong, moderate, and weak biofilm production and nonproduction. Early detection of biofilm production in MRSA could help in preventing treatment failure and better management of S. aureus infections.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3]


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