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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 14  |  Issue : 1  |  Page : 21-24

Distribution of IcaA and IcaB Genes in Biofilm-Producing Methicillin-Resistance 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 Submission15-Dec-2021
Date of Decision11-Jan-2021
Date of Acceptance13-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
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ajprhc.ajprhc_16_21

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  Abstract 


Background: Biofilm-producing methicillin-resistance Staphylococcus aureus (MRSA) strains may reduce the penetration rate of antibiotics which leads to treatment failure; therefore, the aim of our study was to assess the presence of icaA and icaB biofilm-producing genes in clinically isolated MRSA strains. Materials and Methods: All pus samples received in the microbiology laboratory were processed as per the standard microbiological procedure for the isolation of S. aureus. Detection of MRSA is done by the cefoxitin disc diffusion method. Phenotypic detection of biofilm in all MRSA strains was accessed by tissue culture plate (TCP) method and polymerase chain reaction as genotypic method was used for detection of icaA and icaB genes. Results: A total of 222 S. aureus were isolated from pus specimens. Out of these, 127 isolates were MRSA (57%). TCP method demonstrated that 68% of MRSA were biofilm producers, on the other hand, 52 (41%) strains showed the presence of icaA gene, and 39 (31%) strains showed the presence of icaB genes in 127 MRSA strains. Conclusion: The detection of biofilm in S. aureus should be considered as essential for better management of diseases caused by biofilm-producing S. aureus strains.

Keywords: Biofilm, icaA gene, icaB gene, methicillin-resistance Staphylococcus aureus, Staphylococcus aureus


How to cite this article:
Malik N, Bisht D, Aggarwal J, Rawat A. Distribution of IcaA and IcaB Genes in Biofilm-Producing Methicillin-Resistance Staphylococcus aureus. Asian J Pharm Res Health Care 2022;14:21-4

How to cite this URL:
Malik N, Bisht D, Aggarwal J, Rawat A. Distribution of IcaA and IcaB Genes in Biofilm-Producing Methicillin-Resistance Staphylococcus aureus. Asian J Pharm Res Health Care [serial online] 2022 [cited 2022 Aug 19];14:21-4. Available from: http://www.ajprhc.com/text.asp?2022/14/1/21/338790




  Introduction Top


The genus Staphylococcus has at least 45 species. The four most frequently encountered species of clinical importance are Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The S. aureus is Gram-positive spherical cocci, usually arranged in grapelike irregular. S. aureus is a member of the normal microbiota of the skin and mucous membranes of humans, therefore, cause suppuration, abscess formation, a variety of pyogenic infections, and even fatal septicemia.[1],[2] S. aureus is one of the most common causes of hospital- and community-acquired infections. The pathogenic S. aureus often hemolyze blood, coagulate plasma, and produce a variety of extracellular enzymes, toxins, and biofilm. S. aureus rapidly develops resistance to many antimicrobial agents, and the classic example is methicillin resistance S. aureus (MRSA), which consequently presents difficult therapeutic problems and biofilm-producing strains make it worse.[3] One of the probable reasons for drug resistance is biofilm production as biofilm is made up of multi-layered cell clusters embedded in a matrix of extracellular polysaccharide (EPS), which provide adherence to the bacteria. EPS of biofilms facilitate greater resistance to the opsonization by antibodies and phagocytosis.[4] Production of biofilm in S. aureus is regulated by the expression of polysaccharide intercellular adhesion proteins. These proteins are encoded by the gene locus icaADBC and mediate cell to cell adhesion, thus facilitate biofilm formation.[5] Infections with biofilm-producing MRSA strains are often chronic and persistent in nature and are the leading cause of morbidity and mortality in health-care settings. Therefore, the present study's aim is to find out the presence of icaA and icaB genes in MRSA and their association with biofilm production.


  Materials and Methods Top


A prospective study was carried out in the Department of Microbiology, Santosh Medical College, Ghaziabad, UP. Approval of the study was obtained from the IEC (Institutional Ethics Committee-SU/R/2021/1844) through the proper channel.

Bacterial isolation

All pus specimens from in and outpatient departments of our tertiary care hospital were included for the present study. All samples received in the microbiology laboratory were processed as per the standard microbiological procedure for the isolation of S. aureus. Five percent Sheep Blood Agar and nutrient agar were used for the isolation of S. aureus, and identification was done using the standard biochemical tests such as:

  1. Gram stain-S. aureus was Gram-positive cocci arranged predominantly in clusters;
  2. Three percent catalase test-positive;
  3. Slide/tube coagulase-positive;
  4. Mannitol fermentation-positive;
  5. DNase test-positive.[6]


Methicillin resistance Staphylococcus aureus detection by cefoxitin disc (30 μg) diffusion method

An isolate was considered to be a MRSA strain if the cefoxitin (HiMedia) zone of inhibition was ≤21 mm.[7]

Biofilm detection by tissue culture plate method

The isolates were placed in brain − heart infusion broth (HiMedia) and incubated for 24 h at 37°C. Biofilm production was detected using 96 well microtiter plates. First, 200 μl of 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. The contents of the wells were discarded and removed by tapping the plate after 24 h. Then, 200 μl PBS 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. The plates were allowed to dry before being analyzed using an ELISA plate reader at 570 nm. The reading values are interpreted as:[8]

ODc: Optical density of growth control.

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


Bacterial DNA extraction and detection of icaA and icaB genes

The genomic DNA from each S. aureus isolate was extracted using the DNA Extraction Kit (QIAGEN: QIAprep Spin Miniprep Kit), following the manufacturer's instructions. The icaA and icaB genes were amplified from DNA of MRSA by the polymerase chain reaction (PCR) (Bio-Rad T100) to detect 188 bps and 270 bps amplicons, respectively, with the following program: 94°C, 5 min, followed by 34 cycles of (94°C, 30 s, 53°C, 30 s, and 72°C, 30 s) ending with 72°C for 1 min. Tm for IcaA (188 bp) was 55°C and for IcaB (270 bps) was 50°C. After amplification of PCR products was run on 1.5% gel agarose for product separation on electrophoresis, and then, PCR products were visualized under ultraviolet transilluminator (Bio-Rad).[9]


  Results Top


A total of 222 S. aureus were isolated from pus specimens which were received from various clinical departments. Out of these isolates, 127 were MRSA (57%). All 127 MRSA isolates were further tested for their biofilm production by tissue culture plate (TCP) phenotypic method. icaA and icaB genes were detected by genotypic method. TCP method demonstrated that 68% of MRSA were biofilm producers. Among them, majority 72%, were strong film producers, 22% isolates were moderate, and 6% were weak biofilm producers. Out of 127 MRSA strains, 52 (41%) strains showed the presence of icaA gene. Out of these 52 strains, 45 strains showed the presence of icaA genes in 86 TCP-positive strains (biofilm producers), and 07 strains showed the presence of icaA genes in 41 TCP-negative strains (nonbiofilm producers), on the other hand, 39 (31%) strains showed the presence of icaB genes in 127 MRSA strains. Out of these 39 strains, 28 strains showed the presence of icaB genes in 86 TCP-positive strains, and 11 strains showed the presence of icaB genes in 41 TCP-negative strains as described in [Table 1],[Table 2],[Table 3].
Table 1: Biofilm detection by tissue culture plate method

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Table 2: Presence of icaA gene in biofilm-producing and nonbiofilm-producing strains by tissue culture plate

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Table 3: Presence of icaB gene in biofilm-producing and nonbiofilm-producing strains by tissue culture plate

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  Discussion Top


Infections with MRSA followed by biofilm formation become a major problem, as it is difficult to treat. S. aureus's capacity of biofilm production has a direct association with their virulence and multidrug-resistance status. Our study revealed that 57% S. aureus strains were MRSA, identified by phenotypic method. A study has been documented on the prevalence of MRSA isolates which varies (13%–74%) among the different geographic regions to different hospitals.[10] The ability of MRSA to produce biofilm helps the bacteria to survive in hostile environments and is considered to be responsible for persistent or chronic infections.[11] The TCP test is an economical and convenient quantitative method for the identification of biofilm-producing bacteria because it provides optimal culture conditions for biofilm formation. This technique detects extrapolysaccharide production in vitro with the help of spectrophotometric measurements, and further is able to categorize bacteria into strong, moderate, and weak biofilm producers.[12] The current study showed that 68% of MRSA isolates were biofilm producers by TCP method. Almost similar findings (65% biofilm production) were published by Hassan et al.[13] whereas Mathur et al.[14] documented 54% biofilm production and Abdel Halim et al.[15] reported 43% biofilm production by TCP method in MRSA which were less compared to our findings. The present study revealed that icaA gene was present in 41% of MRSA strains which were mostly biofilm-producing strains. In concordance to our data, Omidi et al.[16] concluded that icaA gene was frequently detected in strong biofilm producers which directly indicates the involvement of icaA gene in biofilm production in S. aureus. Researchers from Brazil[17] detected the icaA gene in 52% of the isolates, whereas Gowrishankar et al.[18] documented 84% of S. aureus isolates had icaA gene. Bissong and Ateba[19] detected icaA, icaB, icaC, and icaD genes in 75% of S. aureus isolates, on the other hand, our study found 54% isolates had icaA and icaB genes which reflect ica genes association in biofilm production. Our study detected icaB gene in 31% MRSA isolates, whereas Ghasemian et al.[20] reported high occurrence (64%) of icaB gene than our study. Our findings showed a decent presence of icaA and B genes in MRSA isolates, but the presence of the biofilm-related genes did not always associate with biofilm production in vitro or in vivo; therefore, phenotypic and genotypic characterization is important to understand the proper mechanism and their regulation for the better management of the disease caused by MRSA.[21]


  Conclusion Top


Biofilm-producing strains of S. aureus, especially MRSA, have showed direct influence on its virulence, and these stains are even more resistant to antibiotics and pathogenic. The present study demonstrated that there was a significant association between phenotypic biofilm production and the presence of icaA and B genes in MRSA strains; therefore, we suggest that biofilm detection by phenotypic methods in S. aureus should be performed on a regular basis followed by genotypic detection to understand the different adhesion mechanisms as it could also be beneficial in the development of new preventive and therapeutic measures. Early detection of biofilm production by phenotypic and genotypic methods in MRSA could help in preventing treatment failure and better management of Staphylococcal aureus infections.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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