Detection of Biofilm Production and Antibiotic Susceptibility Pattern among Clinically Isolated Staphylococcus aureus

Aim The increasing antibiotic resistance and the ability to form biofilms in medical devices have become the leading cause of severe infections associated with Staphylococcus aureus (S. aureus). Since the bacteria living in biofilms can exhibit 10- to 1,000-fold increase in antibiotic resistance and implicate chronic infectious diseases, the detection of S. aureus ability to form biofilms is of great importance for managing, minimizing, and effectively treating infections caused by it. This study aimed to compare the tube and tissue culture methods to detect biofilm production and antibiotic susceptibility in MRSA and MSSA. Materials and Methods The S. aureus isolates were identified by the examination of the colony morphology, Gram staining, and various biochemical tests. Antimicrobial susceptibility testing of all isolates was performed by the modified Kirby–Bauer disc diffusion method as recommended by CLSI guidelines. MRSA screening was performed phenotypically using a cefoxitin disc (30 µg). Isolates were tested for inducible resistance using the D-test, and two phenotypic methods detected biofilm formation. Results Among 982 nonrepeated clinical specimens, S. aureus was isolated from 103 (10.48%). Among 103 clinical isolates of S. aureus, 54 (52.42%) isolates were MRSA, and 49 (47.57%) were MSSA. Among 54 MRSA isolates, the inducible MLSB phenotype was observed in 23/54 (42.59%) with a positive D-test. By TCP method, 26 (48.1%) MRSA isolates were strong biofilm producers, whereas, among all MSSA isolates, only 6 (12.2%) were strong biofilm producers. Conclusion MRSA showed strong biofilm production in comparison with MSSA. The TCP method is a recommended reliable method to detect the biofilm among S. aureus isolates, and the TM method could be useful for the screening of biofilm production in S. aureus in the routine clinical laboratory.


Introduction
Staphylococcus aureus, a virulent human pathogen, is the most known cause of community and nosocomial infections.Almost 41% of the general population carries S. aureus, colonizing the upper respiratory tract asymptomatically as a human microbiota, which might serve as a source for invasive infections [1], resulting in minor to severe systemic infections such as pulmonary infections, and infective endocarditis, leading to treatment failures and death [2,3].
With the improper use of multiple drugs and development of resistance to the drugs, their ability to form bioflm, S. aureus is capable of causing diverse infections not responsive to the usual therapy [4].Bacteria living as a bioflm generally show increased resistance to antibiotics and can evade the host immune system acting in the complement cascade, impairing deposition of C3b, key complement component, and C3b opsonization, complicating the effectiveness of antimicrobial therapy and clearance from the body [5].
Te S. aureus isolates resistant to methicillin were frst described in 1961, shortly after the introduction of methicillin in clinical practice; however, MRSA strains were present even before the introduction of methicillin, which was thought to be due to the use of penicillin previously [6].Furthermore, the irrational use of conventionally available antibiotics and ability of forming bioflms has increased antibiotic resistance among S. aureus and MRSA isolates often show multidrug resistance (MDR), resistance to β-lactam antibiotics and tetracyclines, macrolide, chloramphenicol, and fuoroquinolones commonly used in the treatment and management of S. aureus infection [7].
Te structured community of bacterial cells enclosed in a self-produced polymeric matrix is defned as a bioflm that is adherent to an inert or living surface [8].A bioflm in S. aureus is generally associated with the expression of polysaccharide intercellular adhesion (PIA) protein, which is a poly-β(1-6)-N-acetylglucosamine (PNAG).PIA is responsible for cell aggregation and cell-to-cell adhesion and is encoded by ica operon (icaADBC) [9].Te increasing use of catheters, endotracheal tubes, prosthetic devices, etc. has increased the risk of bacterial infections like staphylococcal infections associated with bioflm formation in medical devices, which could be the reason for increased morbidity, mortality, and socioeconomic burden [10].Hence, the detection and diferentiation of S. aureus and its ability to form a bioflm in such devices are of great importance for managing, minimizing, and efectively treating such infections [11].Various phenotypic methods such as the tissue culture plate method (TCP), Congo-red agar method (CRA), tube method (TM), and electron microscopy are available for the detection of the bioflm.However, the TCP is quantitative and considered the gold standard for bioflm detection, whereas CRA and TM are qualitative methods [12].

Materials and Methods
Tis laboratory-based cross-sectional study was conducted from July 2020 to December 2020 at the Department of Microbiology, Global Hospital, Lalitpur, Nepal.

Inclusion Criteria.
Samples collected from patients of all age groups and genders visiting the hospital during the study period with suspected infections were included in the study.

Exclusion Criteria.
Repeated samples, samples collected after antibiotic therapy, and samples showing signs of contamination were excluded from the study.

Sample Processing and Identifcation of S. aureus.
A total of 982 nonrepeated clinical specimens collected from July 2020 to December 2020 were included in the study.All the samples were processed in the microbiology laboratory following the standard microbiological procedures.Initially, all the samples were streaked on blood agar and mannitol salt agar plates and incubated aerobically at 37 °C for 24 hr.Te isolates were frst identifed as staphylococcal strains depending on the colony morphology and Gram staining.Te yellow-colored colonies on mannitol salt agar and cream to golden yellow colonies with β or weak hemolysis on blood agar were subcultured on nutrient agar (NA) and incubated aerobically at 37 °C for 24 hr.After incubation, the isolated colonies from NA plates were used for biochemical tests including catalase, coagulase (slide and tube coagulase test), and DNase tests for the identifcation of S. aureus isolates.

Phenotypic Detection of MRSA.
All isolates of S. aureus were screened for MRSA phenotypically using a cefoxitin disc (30 µg) (HiMedia Pvt. Ltd.India).Isolates were lawn cultured on MHA, a cefoxitin disc was placed, and the plates were incubated aerobically at 37 °C for 24 hr.S. aureus yielding a zone diameter less than 21 mm with cefoxitin disc were phenotypically confrmed as MRSA [14].

Phenotypic Detection of Inducible Clindamycin Resistance (iMLSB Phenotypes
). Te S. aureus isolates showing resistance to erythromycin were tested for inducible resistance to clindamycin.In addition, isolates were tested for inducible resistance using the D-test as per the CLSI guidelines.In brief, 0.5 McFarland standard bacterial suspension was lawn cultured on MHA plates on which the erythromycin disc (15 µg) and clindamycin disc (2 µg) were placed 15 mm apart from edge to edge.Te plates were incubated aerobically at 37 °C for 24 hr; if the isolate was resistant to erythromycin (zone diameter ≤13 mm) and susceptible to clindamycin (zone diameter ≥21 mm) with a D-shaped zone around clindamycin, it was considered positive for inducible clindamycin resistance [3] (Figure 1).

Bioflm Formation Assay.
Bioflm formation was detected by in vitro methods: Tube method (TM) and tissue culture method (TCP).Strong bioflm-producing S. epidermidis strain ATCC 35984 was used as a positive control in both the tests performed.
2.8.Tube Method.Bioflm detection using the tube method was performed as per Christensen et al. [15].Briefy, a loop full of inoculum from overnight culture plates was inoculated in 5 ml trypticase soy broth (TSB) with 1% glucose and incubated overnight at 37 °C.Te tubes were emptied 2 Journal of Pathogens following incubation, washed with phosphate bufer saline (PBS) pH 7.2, air-dried, and stained with 0.1% crystal violet followed by washing with deionized water to remove excess stain.Te tubes were air-dried and observed visually for bioflm production.Bioflm production was indicated by the formation of a visible flm lining the wall and bottom of the tube while ring formation at the liquid air interface was not considered bioflm production.Te results were categorized in reference to the control strain as weak/nonproducers, moderate, and strong bioflm producers, depending upon the intensity of the flm observed visually (Figure 2).
2.9.Tissue Culture Plate (TCP) Method.Bioflm detection using the TCP method was performed as per Christensen et al. [16].Isolates of S. aureus from the new culture were inoculated in 2 ml TSB with 1% glucose and incubated overnight at 37 °C. 1 : 100 dilution was prepared with fresh TSB medium, and 200 µl of dilution was inoculated into individual wells of sterile, fat-bottom polystyrene tissue culture plates.At the same time, S. epidermidis strain ATCC 35984 was also processed simultaneously, which served as a positive control, and un-inoculated wells, which served as a negative control.Te plates were incubated overnight at 37 °C.Following incubation, the contents of each well were removed by pipetting and tapping the plates gently followed by washing with 200 µl PBS (pH 7.

Discussion
Staphylococcus aureus, the common cause of community and healthcare-associated infections, is capable of causing multiple acute and chronic infections such as pulmonary infections and infective endocarditis.Colonization of S. aureus as bioflms in nasopharynx serves as a reservoir for local and invasive diseases.A recent study suggested S. aureus and Streptococcus pneumoniae can cocolonize the nasopharynx forming the dual species bioflms and S. pneumoniae modulates S. aureus bioflm dispersion and transition from colonization to invasive disease.Additionally, physiological changes in response to certain infections like infuenza A virus (IAV) promote the dispersal of bacteria from the bioflms and disseminate from colonized nasal  tissue to the lungs [18].Similarly, the ability of S. aureus to form bioflms in indwelling medical devices such as catheters and prostheses and increasing antibiotic resistance has led to the severe infections associated with S. aureus not responsive to usual therapy.Terefore, timely and early detection of bioflm production among S. aureus highlights the vital steps to prevent, manage, and adequately treat infections caused by it [2].Furthermore, the efective and reliable diagnostic method is essential in healthcare settings for the timely management of bioflm-associated staphylococcal infections [19].Terefore in this study, we examined bioflm formation by two commonly and routinely used phenotypic methods of in vitro bioflm detection.In this study, phenotypic methods tested 103 clinical isolates of S. aureus isolated from diferent clinical samples for antibiotic sensitivity and bioflm production.Among 103 clinical isolates of S. aureus, 54 (52.42%) isolates were MRSA, and 49 (47.57%) were MSSA.Our study showed a slightly higher incidence of MRSA isolates compared to the previously published reports: 43.1%, 45.9%, and 47.4% of MRSA isolates from Nepal [20][21][22].Te incidence of MRSA seems to be increasing in Nepal as the recently published reports by Sapkota et al. (70.6%) [23] and Dhungel et al. (87.2%) [24] have reported higher cases of MRSA than in our study.
In our study, we found that 94.4% of MRSA isolates were resistant to ceftazidime, which is in accordance with the study performed by Hussaini et al., where they found that 100% of MRSA isolates were resistant to ceftazidime [25].Similarly, the higher rate of resistance among MRSA isolates was with ampicillin-clavulanate (89%), followed by ofoxacin (77.8%).Resistance to cotrimoxazole among MRSA isolates was found to be 74%, which is higher as compared to 63.2% reported by Belbase et al. [20] and 55.9% reported by Manandhar et al. [2].Furthermore, in contrast to 32.7% erythromycin resistance noted by Ansari et al. [21] and 55.3% noted by Belbase et al. [20], we reported a higher rate (64.8%) of resistance to erythromycin among MRSA isolates.In contrast to the previously published studies, we found that 29.7% of MRSA were resistant to vancomycin [20].In accordance with our fndings, a study performed by Goswami et al. in India reported that 45.83% of S. aureus isolates were resistant to vancomycin.Te study also suggested that the resistance of S. aureus to vancomycin should be interpreted cautiously as it could be a false-positive result due to the contamination with bacteria like Acinetobacter and also mentioned that resistance to vancomycin among MRSA isolates is an alarming situation [26].
In our study, higher rates of multidrug resistance were found among MRSA isolates due to the various risk factors contributing to resistance in developing countries like Nepal.Te lack of awareness among the public about the proper use of antibiotics, availability of antibiotics without prescription and prescription by unauthorized personnel, self-medication by the patient without consulting the doctor, improper dosage of antibiotics, and incomplete courses of antibiotics use, and the lack of proper facilities in the laboratories to detect antibiotic resistance could explain the increasing rate of antibiotic resistance among clinical isolates in Nepal [21].Since the increasing rate of antibiotic resistance and bioflm-forming ability among S. aureus is worrisome, possible alternative treatments are of great concern.Recently, Sempere et al. [27] have identifed that antioxidants such as N-acetyl-L-cysteine (NAC) and cysteamine (Cys) can be the promising drug candidates against individual or mixed S. aureus bioflms.Additionally, the higher sensitivity of linezolid, amikacin, and clindamycin among MRSA and MSSA isolates in this study indicates these antibiotics could be used as an option for the preliminary treatment of Staphylococcal infections.
Furthermore, we detected inducible clindamycin resistance in 40.77% of isolates, which is signifcantly higher in comparison with previously published reports [28][29][30], and in accordance with the previously published reports from Nepal [29,30], it was higher among MRSA isolates (42.5%) as compared to MSSA isolates (38.7%).With the emergence of resistance among S. aureus to multiple antibiotics, the use of reserve drugs (like the MLSB family) is being opted for the management of S. aureus infection.However, among the MLSB family, clindamycin is the better choice due to its lower side efects and better tissue penetration capability.And during clindamycin therapy, iMLSB strains can gradually develop constitutive or inducible clindamycin resistance, leading to treatment failure in some patients.Hence, to minimize treatment failures, detecting such  resistant phenotypes is essential in routine practice [31].Terefore, these fndings indicate that the D-test is vital in routine laboratories for the preliminary identifcation of inducible clindamycin resistance, which could efectively manage and minimize treatment failures that are likely to occur.In our study, bioflm formation was detected using two diferent phenotypic assays, i.e., TM and TCP.As mentioned by Hassan et al., the TCP method is the gold standard method for the detection of bioflm [32,33].Since there is the use of multiple controls and the experiments being performed in triplets, TCP provides better assessment of S. aureus bioflms.Similarly, Manandhar et al. mentioned that the best correlation was shown by the TCP method with the presence of icaAD genes [2]; hence, we considered it as a standard method for the interpretation of our results.With the TCP method, strong bioflm production was signifcantly higher among MRSA isolates (48.1%) in comparison with MSSA isolates (12.2%), which is in accordance with the fndings of Manandhar et al. [2].Similarly, Piechota et al. in Poland also found a higher rate of bioflm formation among MRSA isolates than MSSA isolates [7].A higher rate of bioflm formation among MRSA isolates indicates bioflm detection in routine laboratories for managing antibiotic resistance and minimization of treatment failure.
Due to resource and time limitations, we could not perform the molecular testing of methicillin resistance and bioflm formation among S. aureus isolates, and the minimum inhibitory concentration test for vancomycin and the study was limited to a single center only.

. Conclusion
Te present result demonstrates that MRSA and MSSA showed bioflm production; however, MRSA showed strong bioflm production compared to MSSA.Bioflm production results in resistance to antibiotics in S. aureus.Early detection of bioflm production in clinically isolated S. aureus mitigates the use of resistant antibiotics in patients.TCP is a recommended reliable method to detect bioflm production among S. aureus, additionally TM could be useful method in routine screening of bioflm production among S. aureus isolates in a resource-limited settings.Terefore, we recommend screening the bioflm production in S. aureus in the routine clinical laboratory.

Figure 1 :
Figure 1: Phenotypic detection of inducible clindamycin resistance by the D-test.

Table 1 :
Distribution of OD value in bioflm formation.

Table 2 :
Distribution of bacterial isolates.

Table 3 :
Antibiotic susceptibility pattern of bacterial isolates.

Table 4 :
Inducible clindamycin resistance in MRSA and MSSA.