Iranian Journal of Medical Microbiology

Biofilm is the aggregation of bacteria embedded in an extracellular matrix that can create on both abiotic and biotic surfaces (1). This layer provides ideal conditions for bacteria to grow in unstable environments. The most important features of biofilm are bacterial survival in extreme environmental conditions, role in pathogenicity and creating chronic diseases, strengthening drug resistance through antibiotic impermeability in the polymeric matrix, facilitate gene transfer through conjugation and increase genetic mutations due to these bacterial connections, development of new genotypic strains by mutation within the biofilm and activation of genes that they are responsible for bacterial virulence (2, 3). S. aureus is one of the pathogens that can cause disease in humans and animals (4). This bacterium is the cause of many diseases in humans, included skin infections, acute and invasive infections such as pneumonia, soft tissue and bone infections, heart valve infections and lethal sepsis. It is one of the most important causes of bacteremia and is responsible for 20-40% of deaths (5, 6). In addition to resistance, this bacterium has the ability to produce various pathogenic factors such as toxins, surface antigens, extracellular enzymes and biofilm production (6, 7).
In S. aureus, the pathogenicity of the biofilm is very important. The presence of this layer is encoded by a structural operon called intra cellular adhesin (ica), which has different gene locus including icaA, icaD, icaB and icaD (8,9, 10). The icaA gene is a primary inducer for start biofilm production. It is activated by the presence of UDP-N-acetyl glucosamine and is the only ica gene that has transferase properties. The IcaD protein is a messenger (chaperone) for other genes in this locus that helps the icaA gene, activates certain enzymes to express the icaC and icaD genes. When the icaA and icaD genes start cooperation, biofilm production increases 20 times (9, 10). The icaC gene communicates between the inside and outside of the bacterial cytoplasmic membrane, that it communicates the IcaD on the inside of the cytoplasmic membrane to the IcaB on the outside. The IcaB is the extracellular protein and cause the superficial association of the bacterium with intercellular adhesion polysaccharide (IAP). One of the factors that increases the expression of this operon is the relationship between IcaB and IPA, that more correlation leads to more biofilm production (11).
Biofilms in hospital environments, that are considered as a reservoir of infection transmission, responsible for causing 65% of nosocomial infections (12). Biofilm binds bacteria to surfaces and other hospital instruments and in this way, to provide the ground for infection in hospitalized people and users of these medical devices (13). In order to prevent the formation of biofilm and subsequent microbial resistance, increased hospitalization, increased costs, and increased mortality (especially in antibiotic-resistant S. aureus); proper use of antibiotics (under the supervision) as well as arbitrarily use of drugs and antimicrobial compounds, regular evaluation of resistance and expression of related genes at the hospital can greatly reduce the incidence of these problems.
Due to the high prevalence of nosocomial infections caused by S. aureus and also the spread of factors that increase antibiotic resistance, the present study aimed to evaluate the genotypic and phenotypic biofilm formation by S. aureus isolated from clinical samples and their association with antimicrobial resistance.

 

This descriptive cross-sectional study from Dec 2019 to Sep 2019 was conducted in the School of Paramedical Sciences, AJA University of Medical Sciences. The present study investigated by the ethics committee of the AJA University of Medical Sciences and approved with the code IR.AJAUMS.REC.1397.087. Using the formula for calculating the sample size and according to the results of similar studies (3, 10), 200 samples were calculated.
A total of 200 clinical samples from 63 patients admitted to different wards of AJA hospitals in Tehran were collected. Samples included blood (63 samples), urine (63), catheter (23 samples), discharge (17), ulcers (16 samples), sputum (12 samples), and nasal swabs (6 samples) which were referred to the paramedical faculty of the AJA University of Medical Sciences under sterile conditions. Inclusion criteria to study were included hospitalization in different wards of the hospital and long-term treatment with various antibiotics, and exclusion criteria were included outpatient treatment (no hospitalization) and no long-term use of antibiotics.
The samples were first cultured on blood Agar medium and incubated for 24h at 37°C. After observing the colonies, standard biochemical tests were performed to confirm the isolates including catalase test, Gram-staining, and then bacterial culture on mannitol salt agar media (mannitol fermentation test), Baird-Parker agar (formation of black colonies with clear and opaque halos), DNase and coagulase tests (14).

Antimicrobial Susceptibility Test
This test performed by Müller–Hinton agar (MHA) using the standard antibiotic disk including penicillin (10 IU), ciprofloxacin (5 μg), chloramphenicol (30 μg), gentamicin (10 μg), doxycycline (30 μg), Tetracycline (30 μg), clindamycin (2 μg), cotrimoxazole (25 μg), ampicillin (15 μg), erythromycin (15 μg), cephalothin (30 μg) and cefoxitin (30 μg), purchased from HiMedia company )Mumbai, India(. (10, 14). The isolates were further screened for methicillin resistance by the cefoxitin disk (15). In these tests, S. aureus ATCC 33591 was used as a positive control. The results exegesis conducted using Clinical and Laboratory Standards Institute guidelines (16).

Phenotypic Evaluation of Biofilm Producing Strains
Congo red and a modified microtiter plate (MTP) assay were used to identify biofilm-producing strains. Confirmed isolates were cultured on brain heart infusion (BHI) agar medium containing 0.8 g/L Congo red and 36 g/L sucrose. After 24h of incubation at 37°C, black colonies as strong biofilms, dark red colonies as weak biofilms and light red colonies as negative biofilm strains were considered (17).
To quantitatively evaluate the production of biofilm by MTP assay, from the samples enriched on trypticase soy broth (TSB) medium, turbidity equivalent to 0.5 McFarland was prepared and then 200 μL of each suspension was transferred to wells of 96-well polyester microplate and incubated for 20h at 37°C. Then, the wells washed (4 times) using phosphate buffered saline (PBS) and dried completely. Next, the wells were stained with crystal violet dye (1%) for 15 minutes and to remove the dye from the bacterial wall, 100 μL of a mixture of isopropyl alcohol 10% and ethanol 70% was added to each well. Finally, the light absorption of each well at 570 nm was investigated using an ELISA reader. Finally, the optical density (OD) of the wells were measured using an ELISA reader at a wavelength of 570 nm. The test was conducted in triplicate. Exegesis of results were performed as per the criteria explained (18) and the bacteria were divided into weak (non-producer), moderate and strong biofilm producers. S. aureus ATCC 25923 was used as positive control, for the biofilm assay.

DNA Extraction
DNA extraction was performed by the boiling technique using lysis buffer (1% Triton x100, 0.5% Tween 20, Tris 10 mmol with pH: 8 and EDTA 1 mmol) (19).

Molecular Confirmation of S. aureus Isolates
Amplifying the thermonuclease gene by polymerase chain reaction (PCR) was used for molecular confirmation of isolates (Table 1). For this test, distilled water and S. aureus ATCC 33591 were utilized as negative and positive controls, respectively. The final volume of each reaction was considered to be 20 μL and the PCR reaction temperature program is shown in table 2.  The products of PCR were evaluated using 1% agarose gel and UV transilluminator. Samples with 279 bp band were considered as S. aureus (20).

Molecular Identification of Biofilm-Producing Strains
Amplification of icaABCD operon genes (with icaA, icaD, icaC and icaB genes) was used to identify biofilm-producing strains by PCR (Table 1). Distilled water and S. aureus ATCC 25923 were used as negative and positive controls, respectively, for this test. The final volume of each reaction was considered to be 20 μL and the PCR reaction temperature program of this operon is shown in Table 2. The products of PCR were evaluated using 1% agarose gel and UV transilluminator. The size of products of the genes are shown in Table 1 (21).

Table 1. Primers used in the study

Table 2. PCR reaction program (30 cycles)

Statistical Analysis
Data are analyzed by SPSS 20 (SPSS Inc., Chicago, IL., USA). X² (Chi-square) test was utilized for data analysis. P-value<0.05 was considered statistically significant.