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ORIGINAL ARTICLE
Year : 2022  |  Volume : 8  |  Issue : 1  |  Page : 56-61

Biofilm Formation in Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Strains


Department of Microbiology, Vardhman Mahavir Medical College and Safdarjung Hospital, Delhi, India

Date of Submission10-Feb-2021
Date of Decision18-Jun-2021
Date of Acceptance28-Jan-2022
Date of Web Publication14-Feb-2022

Correspondence Address:
Dr. Manisha Jain
Department of Microbiology, Vardhman Mahavir Medical College and Safdarjung Hospital, Delhi-110029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mamcjms.mamcjms_10_21

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  Abstract 


Background: The combination of methicillin resistance (MR) and the ability of biofilm formation of Staphylococcus aureus (SA) makes treatment difficult. Biofilm is formed by distinct genetic mechanisms in MRSA and methicillin-sensitive SA (MSSA), and hence, there is difference in the prevalence of biofilms in them. This study investigated the biofilm production in SA and analyzed its correlation with MR. Materials and methods: A total of 261 consecutive Staphylococcus aureus isolated from various clinical samples from January to April 2019 were included in the study. Antibiotic sensitivity was carried out as per Clinical and Laboratory Standards Institute guidelines and cefoxitin disk was used for screening for MR. Total of 147 MSSA and 114 MRSA were taken for further processing. Biofilm formation was determined by tube and microtiter plate methods in all the isolates. The data were analyzed for statistical significance using Microsoft excel software. Results: Resistance to erythromycin, clindamycin, gentamicin, cotrimoxazole, and inducible clindamycin resistance was significantly higher in all the MR Staphylococcus strain including biofilm forming MRSA strains. The biofilm formation was significantly higher in MSSA isolates by both the tube (78.2%) and microtiter plate methods (64.9%). Discussion: Although the antimicrobial resistance was higher in MRSA, the ability to form biofilms was significantly higher in MSSA. Biofilms of MSSA are usually less prevalent than MRSA probably because of the distinct genetic mechanisms involved in the formation of biofilms. The higher biofilm forming ability of MSSA in our study highlights the need for determination of other genes involved in biofilm formation and virulence mechanisms in SA.

Keywords: Biofilm, MRSA, MSSA


How to cite this article:
Singhal A, Jain M, Gaind R. Biofilm Formation in Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Strains. MAMC J Med Sci 2022;8:56-61

How to cite this URL:
Singhal A, Jain M, Gaind R. Biofilm Formation in Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Strains. MAMC J Med Sci [serial online] 2022 [cited 2022 May 24];8:56-61. Available from: https://www.mamcjms.in/text.asp?2022/8/1/56/337679




  Introduction Top


Staphylococcus aureus (SA) is one of the most frequently isolated pathogens from various clinical samples. The organism is difficult to treat even after adequate and prolonged antibiotic therapy, suggesting that SA is able to develop specific strategies for adapting to diverse environmental conditions. The biofilms are recalcitrant to antibiotic therapy and pose a serious burden drastically increasing the treatment cost and morbidity of the patient.[1],[2],[3],[4] Biofilms also increase the opportunity for the transfer the resistance genes between bacteria.

Biofilm are formed by distinct genetic mechanisms in both MSSA and MRSA. Biofilm of MSSA is formed by exopolysaccharide polysaccharide intercellular adhesin (PIA) the virulence factor coded icaADBC gene. So the mechanism of biofilm formation in MSSA is ica-dependent also known as PIA-dependent, whereas in MRSA it is formed in ica-independent manner by surface proteins. The mecA gene present in MRSA increases the biofilm formation by inactivating the accessory gene regulator quorum-sensing system which also regulates the virulence of SA.[5],[6] Carriage of SCCmec elements in MRSA suppresses colony spreading activity, reduces expression of the chromosomally encoded PSMa, and thus attenuates virulence.[5],[6],[7] Many studies have found the prevalence of biofilm formation to be higher in MRSA because of the genetic mechanisms involved. Few studies have observed the prevalence of biofilms to be higher in MSSA.

Infections caused by combination of biofilm and MR would pose a major threat to patients. Theoretically infections caused by MSSA are clinically observed as more easily treatable. One of the major reasons for persistence of staphylococcal infection is formation of biofilms, and if the biofilms are present more in MSSA, the infections caused by these sensitive strains could also become persistent.

Thus, the present study was aimed to find out the prevalence of biofilm formation in SA strains and correlate the presence of biofilms with resistance to methicillin and other antibiotics.


  Materials and Methods Top


Bacterial strains

The study was conducted over a period of 4 months from January to April 2019 in the department of microbiology in a tertiary care super specialty hospital in northern India. Various clinical samples such as pus, tissue, respiratory specimens, and others were received in the microbiology laboratory. The samples were processed and isolates were identified using conventional microbiologic techniques such as colony morphology, Gram stain, and coagulase test. All Staphylococcus isolates were confirmed by DNase agar and mannitol salt agar. The isolates were stocked at −20°C in glycerol stock solution for biofilm detection.

Antibiotic resistance profiles of the isolates

Antimicrobial susceptibility of the isolates was performed using the disk diffusion method (Kirby–Bauer method) on Muller–Hinton agar plates as described in the Clinical and Laboratory Standards Institute (CLSI) guidelines. The antibiotics that were used for susceptibility testing of Staphylococcus isolates in this study were penicillin (10 units), gentamicin (10 μg), erythromycin (15 μg), clindamycin (2 μg), sulfamethoxazole-trimethoprim (1.25/23.75 μg), vancomycin (30 μg), and linezolid (30 μg). Cefoxitin (30 μg) disk was used as a surrogate marker for MR. SA isolates that tested resistant to cefoxitin by disk diffusion were reported as MRSA in accordance to CLSI guidelines.

Biofilm formation assay

Tissue plate method (TCP)

The isolates were revived on 5% sheep blood agar incubated at 37°C for 18 to 24 hours. After that, bacterial cells were transferred to brain heart infusion broth (BHIB) to prepare cell suspension containing about 108 CFU/mL. Subsequently, 200 μL of this BHIB was inoculated in double replicates to wells of a tissue culture polystyrene 96-well plate. Biofilms were developed for 48 hours at 37°C. After this time, the supernatant was removed and nonadherent bacterial cells were discarded by washing the biofilms thrice with 250 μL of sterile phosphate-buffered saline (pH 7.4). Biofilm was fixed with 200 μL of methanol per well for 15 minutes and stained for 5 minutes with 200 μL of 1% crystal violet per well. After rinsing with distilled water, the plates were air dried. After that, colorant was solved in 95% ethanol to measure absorbance at 490 nm in microplate reader. Positive and negative controls were put in four wells each time. Cutoff was decided and each value above that was considered positive. Then biofilm was divided into mild, moderate, and severe.

Tube method (TM)

A loopful of inoculum was inoculated on 10 mL BHIB in glass tubes. Tubes were incubated aerobically at 37°C ± 1 for 24 hours. The content of the tube was discarded, and tubes were washed twice with 9 mL PBS pH 7.2 and then discarded. For biofilm fixation, 10 mL of freshly prepared sodium acetate (2%) was added to each tube for 10 minutes and then discarded. For biofilm staining, 10 mL crystal violet (0.1%) was then added to each tube, and tubes were left at room temperature for 30 minutes after which the stain was discarded. The washing step was repeated, and tubes were left to dry in an inverted position at room temperature. Biofilm formation was detected by the presence of visible film on the wall and bottom of the tube. The amount of biofilm formation was interpreted according to the results of the control strain and graded visually as absent, mild, moderate, and strong biofilm formation, respectively.

Statistical analysis

The data were entered in excel sheet and comparison between two parameters was analyzed by using Chi-squared test and P-value was calculated for each parameter. P-value less than 0.05 was taken as statistically significant.


  Results Top


The SA strains included in this study were divided into two groups as MSSA (147 strains) and MRSA (114 strains). The demographic characteristics of 147 MSSA and 114 MRSA isolates were evaluated, and P-value was calculated for the statistical significance. There was no significant difference in the age distribution and gender distribution of MRSA and MSSA. The sample distribution between MSSA and MRSA isolates were observed. Pus swab from various sites was the dominant sample (79.5% and 82.4%) in both the cases. Aspirates isolated more of MSSA (17.6%) than MRSA (14%) but none of the difference was statistically significant. The demographic details and sample distribution between the two categories are summarized in [Table 1].
Table 1 Demographic characteristics of study population

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All the SA strains were evaluated for biofilm formation by microtiter plate and by tube method. Out of 147 MSSA strains, 87.7% were biofilm producers by microtiter plate method and 78.2% by tube method. On the contrary, out of 114 MRSA strains, 75.4% were biofilm producers by microtiter plate method and 64.9% were positive by tube method. Based on absorbance values at 490 nm in plate method and subjective reading in tube method, the strains were considered weak, moderate, and strong producers of biofilm, respectively.

By microtiter plate method, the majority of the 114 MRSA strains examined (47.3%) formed mild biofilms. The strong biofilms were formed by only 7.01% of the MRSA strains, whereas 21% of strains were weak producers of biofilm. Nearly 42.8% of MSSA strains formed strong biofilms, whereas 25.8% and 19% of MSSA strains were moderate and weak producers of biofilms. Comparison of biofilm biomass (absorbance at 490 nm) using the statistical test showed that MSSA strains had significantly higher ability of biofilm formation than MRSA strains (P < 0.00001) [Table 2].
Table 2 Biofilm production by microtiter plate and tube method

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The tube method detected positive biofilm production in 129 (87.7%) of MSSA isolates, the moderate positive were 39 (26.5%), and mild positive were 47 (31.9%). As for the MRSA, positive biofilm production was observed in 74 isolates (64.9%), the strong positive was only 5 (4.3%), and 51 (44.7%) were mildly positive [Table 2].

Considering TCP to be the gold standard sensitivity, specificity, and diagnostic accuracy of tube method were 81.8%, 71.7%, and 80.1%, respectively [Table 3]. Chi-squared test was performed to compare the results of biofilm production by TM and TCP, and the results were highly significant (P < 0.001).
Table 3 Comparison between microtiter plate and tube method for biofilm production

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Inducible clindamycin resistance and antibiotic resistance were significantly higher in MRSA when compared with MSSA [Table 4]. Biofilm-producing strains were more resistant to almost all the classes of antibiotics in both MSSA and MRSA showing resistance to gentamicin (14.2, 36.8%), clindamycin (13.6, 48.2%), erythromycin (42.8, 75.4%), cotrimoxazole (18.3, 28%), and ciprofloxacin (95.2, 98.2%). Biofilm nonproducers were comparatively less resistant. All isolates were sensitive to vancomycin and linezolid. The antibiotic resistance pattern of biofilm producers in MSSA and MRSA is given in [Table 5].
Table 4 Antibiotic resistance pattern in MSSA and MRSA (% resistant)

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Table 5 Antibiotic resistance pattern in biofilm forming MSSA and MRSA (% resistant)

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  Discussion Top


The MRSA that is resistant to β-lactam antibiotics causes life-threatening infections. The biofilm has an important role in the pathogenesis of certain bacterial infections such as staphylococcal indwelling device–associated infections, wound infections, chronic urinary tract infections, cystic fibrosis pneumonia, chronic otitis media, chronic rhinosinusitis, periodontitis, and recurrent tonsillitis.[8] SA is a major human pathogen that causes acute to chronic systemic infections which are often refractory to antibiotics leading to treatment failures. Such recalcitrant infections are mainly associated with biofilms formed by S. aureus on indwelling devices such as catheters, central venous catheters, prostheses and others.[9],[10],[11] Early detection of virulent staphylococci therefore warrants one of the most essential steps for prevention, management, and cure of nosocomial infections. The present study was planned to determine the prevalence of biofilm formation in SA and see the association of biofilms with MR.

Biofilm plays an important role in the pathogenesis of staphylococcal infections. When microorganisms are exposed to stress conditions gene expression of biofilm is induced as a stress response. The biofilm causes bacteria to survive in the stress conditions, causes bacterial attachment and colonization on biotic or abiotic surfaces such as prosthetic surfaces, thus leading to their persistence on these prosthetic devices.[12],[13] The treatment of biofilm-embedded bacteria that are not eliminated completely by antimicrobials even at the high doses is tough and irresponsive. The patients whose indwelling device is infected by biofilm producers have higher risk of mortality. Infected implants that cannot be treated by antibiotics have to be removed out of the body to prevent biofilm-related infections.[14]

Prevalence of biofilm formation in SA has been reported to vary between 50% and more than 70% in some studies.[9],[11],[15] There was a much higher prevalence of biofilms (82.4%, 215/261) in the present study when compared with previous studies. Biofilm formation depends on many factors such as environment, availability of nutrients, geographical origin, types of specimen, surface adhesion characteristics, and genetic makeup of the organism.[16] These factors may have contributed to the high prevalence observed in the present study. Biofilms can form on any wound when planktonic bacteria are not eliminated by the immune system of the host or by exogenous antimicrobial agents.[17] Most of the patients before reaching our hospital are already exposed to antimicrobials and usually the nonresponders reach the hospital. Biofilms might have been the cause of failed treatment in wound infections from such patients in our setting.

Adherence property of SA biofilms is associated with the degree of pathogenicity, and the virulence property of the organism. Therefore, the adherence property of biofilm producers was graded as strong, moderate, and weak in this study (TM and TCP). In the present study, 7% of the SA were highly virulent showing strong adherence, whereas 21% were weak biofilm producers. Our results are consistent with studies from Algeria, which showed 4 (8%) strongly adherent, 20 (40%) weakly adherent, and Pooja et al. who showed 6.9% were strong biofilm producers.[18],[19]

In this study, the detection and comparison of biofilm-forming ability were performed using two in vitro methods, TCP and TM, respectively. TCP is traditionally used as a gold standard method, whereas TM is used as a screening test.[11] Though TM is one of the simplest methods used, there is a high probability of subjective errors.[20] In the present study, the prevalence of biofilm formation was similar by both the methods. It was observed that the tube method also showed a diagnostic accuracy of around 80%. As the tube method is less labor intensive and can be performed even on a single isolate, we recommend that this method can be used in nonresolving wound infections to look for biofilm formation.

The virulence of SA, role of MR on biofilm, and relation between virulence and biofilm are interrelated.[13],[14] The mecA gene, which is located in the staphylococcal cassette chromosome enhances virulence of Staphylococcus by causing resistance to multiple antibiotics. Mechanisms of resistance to β-lactam antibiotics are regulated by regulatory genes in the presence of such antibiotics. In the present study also, higher rate of resistance was observed in association with MR toward antimicrobial agents such as erythromycin, cotrimoxazole, and clindamycin as has been reported by other authors.[21],[22],[23]

In addition, resistance of SA to penicillin and ciprofloxacin was high in both MRSA and MSSA. This could be because of the excessive use of these drugs for the treatment of both minor and more serious staphylococcal infections in our setting. All the strains were susceptible to vancomycin and linezolid and they remain as the only options available for multidrug-resistant SA.

Biofilm is produced by distinct mechanisms in MRSA and MSSA. The genes that are essential for biofilm formation are a subset of those involved in the pathogenesis.[18] The significant association between methicillin susceptibility in SA and ica-dependent biofilm formation was first reported when PIA production was found to be essential for biofilm formation by MSSA but not MRSA.[24]

Biofilm of MSSA is formed in ica-dependent manner (PIA-dependent) by PIA that is encoded by icaADBC gene, whereas biofilm of MRSA is formed in ica-independent manner (PIA-independent) by surface proteins containing LPXTG anchoring domain coded by srtA gene.[13],[14] In MRSA, the mecA gene that is responsible for multidrug resistance also increases biofilm production by inactivating the quorum sensing two-component regulatory system.[14],[25],[26]

Because of these genetic mechanisms, biofilms have been observed to be more prevalent in MRSA.[23],[27],[28] In the present study, contrary to findings by other authors, biofilms were more significantly detected in MSSA by both the phenotypic methods.

Further, strong biofilm formation was significantly higher in MSSA strains when compared with the MRSA strains which produced most biofilms in mild category. Traditionally, MSSA are believed to be less pathogenic but that might not hold true if the MSSA strains start producing more biofilms as observed in the present study. As detailed genetic analysis could not be conducted, the factors leading to higher biofilms in MSSA remained undetermined in the present study. The authors feel that other genes involved in biofilm formation in MSSA should be looked into by detailed genetic studies to look for possible reasons for such an increase.

Low-concentration combination therapies may be effective to eradicate biofilm-related staphylococcal infections including those by MSSA if detected early. Screening of biofilm producers along with their antimicrobial susceptibility profile is important, as both biofilm production and antibiotic resistance are independently associated with poor outcome. Selection of an appropriate antimicrobial agent becomes important in these cases to achieve better outcome. Biofilm detection could be included as a routine in nonresponding patients to reduce treatment failure and limit treatment duration especially in MSSA strains.


  Conclusion Top


The high biofilm forming capacity of MRSA and MSSA strains indicates high ability of these strains to persist in hospital environment and increase the risk of chronicity in hospitalized patients. The antimicrobial resistance was higher in MRSA but the ability to form biofilms was significantly higher in MSSA. The higher biofilm forming ability of MSSA in our study highlights the importance of need for detailed genetic investigations for determination of other genes involved in biofilm formation and virulence in SA.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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