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IDENTIFICATION AND DETECTION OF BIOFILM PRODUCING STAPHYLOCOCCUS AUREUS AND ITS ANTIBIOGRAM ACTIVITIES

Objectives: The main aim of this work is to determine the antibiogram profile of biofilm-producing Staphylococcus aureus from various clinical specimens of the patients. Methods: Various bacterial cultures of non-repeated clinical specimens from a total of 3388 patients were determined using standard microbiological and biochemical methods. Results: Out of 3388 only 604 (17.02%) displayed growth positive. A total of 65 (51.58%) S. aureus isolates were recovered, 25 (38.46%) were identified as methicillin-resistant S. aureus (MRSA) by Cefoxitin (30 µ g) disk diffusion technique, of which majority were from pus/wound swab 22 (37.29%). The antibiogram of the isolates was analyzed by Kirby-Bauer disk diffusion technique analyzing Linezolid to be the most effective drug with susceptibility of 100% to both MRSA and methicillin-sensitive S. aureus, followed by vancomycin, tigecycline, and tetracycline. production by tissue culture plate (TCP) and Congo red agar method detected 52 (80%) and 25 (38.46%) as biofilm producers, respectively. TCP identified 2 (3.07%), 7 (10.76%), and 44 (67.69%) as strongly, moderately, and weakly adherent. About 30.7% of MRSA obtained were positive biofilm producers. The minimum inhibitory concentration value of Oxacillin for S. aureus by agar dilution method ranged from 0.025 µ g/mL to 128 µ g/mL. Conclusion: This study shows that biofilm production was more in methicillin-resistant strains and displayed a high degree of resistance to almost all groups of antibiotics.


INTRODUCTION
Staphylococcus aureus, a Gram-positive, round-shaped bacterium about 0.8-1.0 µm in diameter, a member of the family Micrococcaceae, frequently found in the upper respiratory and on the skin [1]. It is also a pathogen armed with virulence factors including pore-forming toxins, superantigens, phagocytosis inhibitors, biofilm-forming capacity, and evasion of the host immune system [2,3].
A biofilm comprises any syntrophic consortium of microorganisms in which a multilayered cell cluster is embedded in the matrix of extracellular polysaccharide (slime) which facilitate the adherence of these organisms to the medial surface and protect them from host immune system and antimicrobial therapy [4]. A Dutch researcher, Antoni Van Leeuwenhoek observed an animalcule first time on the surface of a tooth using a simple microscope which was considered a biofilm discovery. The biofilm formation involves the production of a polysaccharide intracellular adhesion along with several surface proteins including biofilm-associated protein, S. aureus surface protein G, and fibronectin-binding proteins [5,6].
Infections due to multiple drug-resistant strains are becoming more critical due to their capacity to produce biofilm. The incidents of community-acquired and hospital-acquired S. aureus have been augmenting with the increasing emergence of a drug-resistant strain called methicillin-resistant S. aureus (MRSA). The presence of the mecA gene located on the cassette chromosome in S. aureus (SCC mec) is responsible for methicillin-resistant. MRSA has been considered a global public health threat, causing high nosocomial infections in patients' intensive care units (ICUs), leading to a higher mortality rate [7]. Biofilm formation by MRSA has more resistance to the host immune response and more tolerant to antimicrobials. MRSA was first identified in 1961 in England [8]. The percentage of hospital isolation MRSA in the developed countries has increased from 2% in the 1970s to 30% in the 1990s [9].
In the UK 44% of S. aureus isolated from the health-care system are MRSA and in Japan 60-70% of S. aureus are MRSA in inpatients. In Nepal, the prevalence of MRSA shows an increasing trend, 29.1-68%. The MRSA infection rate reported all over Nepal was 43.1%, which has turned out to be a perilous situation in Nepal [10].
It is now well documented that biofilms are notoriously difficult to eradicate and are often resistant to systemic antibiotic therapy [11]. The risk factor contributes to MRSA infection and colonization include excessive use of antibiotics, prolonged hospitalization especially in ICUs, intravascular catheterization, and immune-compromised states [12,13]. This work aims to isolate S. aureus from different clinical specimens for determining its antibiotic susceptibility, and rate of MRSA and methicillin-sensitive S. aureus (MSSA). Further, it helps, to determine biofilm-producing S. aureus to compare its antibiotic susceptibility pattern using different assay techniques.

Screening of MRSA
Cefoxitin (30 µg) using modified Kirby-Bauer disk diffusion method was used for the screening of methicillin resistance S. aureus. The isolated colonies were prepared in nutrient broth suspension matched with turbidity 0.5 McFarland. A sterile cotton swab was taken and left for 10 min for the diffusion of the antibiotic. The plate was incubated at 37°C for 24 h and zone of diameter was measured and MRSA (<21 mm) was confirmed.

Minimum inhibitory concentration (MIC) of oxacillin by Agar dilution method
Agar dilution method following CLSI guidelines (2014) was employed in determining MIC of oxacillin, ranging dilution of oxacillin from 0.125 µg/mL to 128 µg/mL.

Screening of biofilm production
Screening of biofilm was enumerated using two different assay methods; the Congo red agar (CRA) and tissue culture plate (TCP) methods.
In CRA method, S. aureus was inoculated in CRA comprising brain heart infusion (BHI) broth supplemented with 2% sucrose and Congo red. It was incubated at 37°C for 24 h and the representative plate is presented in Fig. 3. The film produced was observed and interpreted; a positive result indicated black color colonies with a dry crystalline consistency.
In the TCP method, the bacterial suspension was grown on tryptic soya broth (TSB) supplemented with 1% glucose and then diluted to 1:100. The 200 µL of this diluted inoculum poured into the wells of sterile flat bottomed 96 well-polystyrene TCPs and 200 µL of TSB supplemented with 1% glucose used as a negative control. The assay plates were covered with an adhesive foil lid which increased biofilm formation by creating an environment with reduced oxygen tension. After incubation, the optical density (OD) of each well was measured using a multi-well plate reader to quantify the growth. The liquid culture from each well was removed by washing each well 3-4 times with deionized water while taking care to preserve the structure of the biofilm located on the bottom of each good assay. The washed plates were incubated at 60°C for at least 60 min. After fixing the biofilm, staining was performed. The observed biofilm could be detected and quantified using various stains. 50 µL 0.1% CV was added to each well and allowed at least 15 min for staining since biofilms were heat-fixed at 60°C. After washing, 30% acetic acid was added to each well and measured the OD 570 [17,18]. The tissue plate culture is shown in Fig. 4.
The OD of the observed biofilm was calculated usitxng the standard protocol mentioned in the literature (Moghadam et al., 2014).

Quality control
The quality of each test was ensured following standard protocol. The temperature of the incubator and refrigerator was monitored every day. The media and reagents after preparation were tested in each batch, labeled properly, and stored in proper condition. The purity of plates of culture and the biochemical test was performed to ensure that the test was completed in aseptic condition. The plates were incubated at 37°C overnight.
It was ensured that only pure culture was used for identification and antibiotic susceptibility testing of organisms. The sterility of CRA was determined by incubating one plate of each batch in an incubator for 24 h.

Statistical analysis
All the raw data obtained were statistically evaluated using a computerbased software program, Statistical Package for the Social Sciences (SPSS) version 20 software packages. A Chi-square test was used to analyze the association between two variables and p<0.05 was considered statistically significant.

RESULTS AND DISCUSSION
Among a total of 3388 samples, 604 (17.82%) were culture positive and the rest 2784 (82.17%) showed no growth as presented in (Fig. 5). Out of total specimens, 2817 were from outpatients, of which 462 (19.6%) showed positive growth and 2355 (82.02%) were culture negative. Furthermore, 571 samples were from the inpatient department, of which 142 (24.8%) were growth positive and the rest 429 (75.13%) showed no growth.

Distribution frequency of S. aureus in diverse clinical samples
The study included different clinical specimens such as blood, invasive devices, pus/wound swab, swab, tissue, urine, and stool. The S. aureus obtained from a total of 604 growth positive specimens were 65 in number, of which the majority were from pus/wound swab as shown in Table 2.
Distribution frequency of S. aureus concerning age and gender of patients Among 65 isolates of S. aureus, 41 (63.07%) and 24 (36.92%) were obtained from male and female patients, respectively. The maximum number of patients infected belonged to the age group of 21-30 years, followed by the age group of 31-40 years. Furthermore, the number of patients infected belonged to the age group 81-90 years as shown in Table 3.

Antibiotic susceptibility pattern of S. aureus
All S. aureus obtained from different clinical samples were tested for antibiotic susceptibility according to CLSI (2014) by modified Kirby-Bauer's Disk diffusion techniques. Linezolid was found to be the most effective drug with susceptibility of 100% toward both MRSA and MSSA represented in Table 4.  Table 5.

Distribution frequency of MRSA and MSSA in outpatients and inpatients
Out of 25 MRSA, 11 of them isolate confined from inpatients and 14 from outpatients. The association between MRSA occurrences in inpatients was statistically significant (p˂0.05), which demonstrated the fact that the chance of finding MRSA in admitted patients was high as compared to the outpatients as shown in Table 6.
Among 14 MRSA isolates obtained from the outpatients, 12 of them were male and two were female. Furthermore, 11 MRSA isolates were from inpatients, of which seven were male and four females. There was no statistical significance as shown in Table 7.

Sensitivity and specificity of CRA method
The TCP method was considered as the gold standard method for this study and was compared with the results from CRA. The sensitivity and specificity of CRA were found to be 58.13% and 76.47%, respectively.

Distribution frequency of biofilm in outpatients and inpatients
Among 65, S. aureus isolates, 16 (88.89%) and 36 (75.59%) were biofilm producers obtained from inpatients and outpatients, respectively. A higher number of biofilm producers was obtained from inpatients but there was no statistical significance as shown in Table 10.

The contrast of antimicrobial resistance pattern among biofilm producer and non-producer
Strong biofilm producer was relatively more resistant to tested antibiotics compared to non-biofilm producer with Linezolid as an exception, which was detected to be the most effective antibiotic to both biofilm producer and non-producer as presented in Table 11.

Correlation between biofilm and MRSA
Microtiter plate assay used for assessing bacterial attachment, that is, biofilm from the MRSA resulted in 30.7% positive biofilm producer whereas, 69.23% were obtained from MSSA. Out of 25 MRSA, 16 (64%) were biofilm producers, and 9 (36%) non-biofilm producers which facilitate no significant association between methicillin-resistant and biofilm production as shown in Table 12.

MIC of oxacillin
The MIC value of oxacillin for S. aureus ranged from 0.025 µg/mL to 128 µg/mL. The 20 (30.76%) of S. aureus were detected to be MRSA out of 65 isolates by agar diffusion method with cutoff value 4 µg/mL, of which 3 (15%) isolates had a MIC of 128 µg/mL (high-level oxacillin resistant). Those isolates, n=5 which were MRSA by cefoxitin disk diffusion method but MSSA by oxacillin in MIC method had MIC value of 2 µg/mL, 1 µg/mL as shown in Table 13.
S. aureus, a common human microbiota, whose emergence of antibioticresistant strain adept of causing minacious health issues [19]. The present study was performed at Kathmandu Model Hospital.
Male patients (63.07%) displayed a higher frequency of S. aureus infection than female patients (36.92%) on the gender-wise distribution of a total of 53 isolates which incident with the findings of (Shahina et al., 2014), 60.65%, 39.35% in male and females, respectively [23]. The maximum number of patients infected belonged to the age group of 21-30 in both genders, which was consistent with the finding of (Bhandari et al., 2019) [24].
In this study, the most effective antibiotic against S. aureus was Linezolid with 100% sensitivity in both MRSA and MSSA which following the study conducted by (Belbase et al., 2017) and (Moghadam et al., 2014) [25,17]. MRSA strains were more resistant to all tested antibiotics than MSSA. The increased resistance of MRSA to a multitude of antibiotics is probably due to irrational and inappropriate use of antimicrobial agents, disregard to hospital infection control policies showing negligible regard to culture susceptibility pattern while administrating antimicrobial agents [26].
The prevalence of MRSA infection was found to be 38.46% agreeing with (Adhikari et al., 2017) but lower, that is, 26.12% in (Pandey et al., 2012) also, higher that is, 69.1% in (Tiwari et al., 2014) [22,27,28]. The variation might be due to variation in antibiotics usage and infection control in various places, patients, and clinical specimens [29].
Although S. aureus occurrence was higher in outpatients, MRSA isolates were significantly associated (p˃0.05) with inpatients (61.1%)    (Ansari et al., 2014), that is, 66.9% and 33.1% as outpatients and inpatients, respectively. This difference could be due to prolonged hospital stay, instrumentation, and other invasive devices, also S. aureus is a major organism causing nosocomial infections.
A wide variety of microorganisms are capable of developing biofilm. Since, there is no standard protocol for biofilm detection, the most widely used assay for evaluation is the microtiter plate assay (Moghadam et al., 2014). In this study, the TCP method and CRA method were used.
CRA, the phenotypic method detected 25 (38.46%) and 40 (61.54%) as biofilm producer and non-producer, respectively, that coincides with the finding of (Mohamed et al., 2016), that is, 56.8% as positive biofilm producer [31]. However, (Mathur et al., 2006), reported a lesser number of biofilm production, which may be due to the imprecision in the identification of moderate biofilm-producing strains by this method (Hassan et al., 2011) [4,32].
In this study, the majority of biofilm-producing bacteria were isolated from pus/wound, followed by invasive devices and tissue [33]. Furthermore, a maximum number of biofilm producers was obtained from inpatients (88.89%) than outpatients (76.59%), which was statistically insignificant.
Higher rates of drug resistance were found among strong biofilmproducing strains in comparison to biofilm non-producing strains. These findings were in favor of results reported by (Belbase et al., 2017).
Due to the protective nature of biofilm, bacteria growing in it are intrinsically resistant to many antibiotics. Positive biofilm producer was 30.7% in the samples obtained from the MRSA and 69.23% obtained from MSSA. There is no statistical association between methicillin-resistant and biofilm production. In a study conducted by (Grinholc et al., 2007), among all tested strains, only 45-47% of MRSA and 66-69% of MSSA strains were to produce biofilm in vitro. It has been reported that some strains, despite the presence of a locus do no produce biofilm [34].
Both cefoxitin disk diffusion method and oxacillin agar dilution method were performed for the detection of MRSA. In this study, 38.46% isolates were found MRSA by cefoxitin disk diffusion method and 30.78% isolates were found MRSA by oxacillin agar dilution method. In a study conducted by (Adhikari et al., 2017), 35.50% and 31.82% were identified as MRSA by cefoxitin disk diffusion method and oxacillin micro-dilution method.
Those isolates with n=5 were MRSA by cefoxitin disk diffusion method but MSSA by oxacillin MIC method had MIC value of 2 µg/mL, 1 µg/mL, respectively, allied with the study conducted by (Adhikari et al., 2017) [27].

CONCLUSION
The prevalence rate of isolation of MRSA from hospitalized patients of Staphylococcus positive cases was found to be high. The pus/wound was the main source of S. aureus and MRSA in hospital settings. The findings of the study on gender-based evaluation displayed a higher frequency of MRSA in male patients compared to female patients between the age group of 20 and 29 years. For the treatment of S. aureus infection including MRSA linezolid (100%) was the drug of choice followed by chloramphenicol (92%), doxycycline (84%), and gentamicin (76%). MRSA strains displayed multidrug-resistant properties and were unusually resistant even to Vancomycin, the drug of choice, which means the emergence of MRSA is dynamic. Hence, reducing this threat