Antibiogram of Biofilm Producing and Non-Producing Community Acquired-Methicillin Resistant Staphylococcus aureus Isolated from Potential Risk Population of Dharan, Nepal CURRENT STATUS: POSTED

Background Staphylococcus aureus is one of the common cause of hospital acquired infection and community acquired infections. Nowadays these organisms became resistant towards variety of drugs. MRSA is the emerging antibiotic resistant bacteria that are resistant to methicillin antibiotic and known to be the infectious pathogen causing severe infection and a cause of fatal mortality.Materials and methodology Altogether 200 nasal swabs and 200 hand swabs were obtained from participants and transported to microbiology lab in cold chain. The samples were swabbed in mannitol salt agar (MSA) containing oxacillin powder of 6mg/L and incubated at 37°C for 24 hrs. Staphylococcus aureus colonies were identified based on growth characteristics on MSA plates (golden yellow colonies), Gram stain and positive results for coagulase tube test and catalase test. The pure isolated MRSA were subjected to antibiotic susceptibility tests and biofilm formation assays. Results From our study the overall prevalence of CA-MRSA was 61.5%. Higher frequency of multi-drug resistant MRSA was isolated. The biofilm producing CA-MRSA were 51.2% which showed high drug resistance and rest (48.7%) were non-biofilm producers. There was significant association in biofilm production with multi-drug resistance (p<0.05). Ciprofloxacin was most sensitive drug against the isolates which was statistically significant (p<0.05). The resistant pattern of biofilm producers reported high ability of multi-drug resistance compared to non-biofilm producers (p<0.05). Microtitre plate method was found to be gold standard over tube and congo red agar method for screening biofilm formation. Surprisingly the emergence of VISA and VRSA strains were significantly reported from our study. The prevalence of VISA and VRSA among CA-MRSA was found to be 49.5% and 40.6% respectively among the isolates which indicates the failure of Vancomycin drug in clinical therapy. Conclusions The prevalence of CA-MRSA was found more in barbers followed by beauticians and municipal waste workers in comparison to healthy controls. This study reported the higher carriage of CA-MRSA in potential risk population along with emergence of VISA and VRSA strains. Improvement in workers the isolated MRSA were 29 out of which 16 (32%) were from hand swab and 13 (26%) were from nasal swab. In healthy population isolated MRSA were 28 out of which 14 (28%) were from hand swab and 14 (28%) were from nasal swab. The frequency of isolated MRSA was reported more from the barbers followed by beauticians and municipal waste workers. This study reported the carriage of CA-MRSA higher in potential risk population. The prevalence of MRSA in hand and nasal of Barber’s population was statistically significant (p<0.05).

37ºC for 24 hours for biofilm production. The unbound cell was discarded and washes several times by PBS (pH-7.2). 125μl of 0.1% crystal-violet solution was added and left for 10-15 minutes incubation. The plate was washed and left inverted for dry. The quantitative determination was performed by solubilizing the biofilm by adding 125μl of 30% acetic acid to each well and incubated the plate for 10-15 minutes at room temperature and transfer to another microtitre plates and reading the absorbance at 570 nm by ELISA plate reader. The interpretation of biofilm production was done according to the criteria of Stepanovic et al. (2007) [10]. Interpretation is made on optical density (OD) of test wells. The optical density (OD s ) of each strain was obtained by the arithmetic mean of the absorbance of three wells and this value was compared with the mean absorbance of negative controls (OD nc ). The following classification was used for the determination of biofilm formation: no biofilm production (OD s ≤OD nc ), weak biofilm production (OD nc <OD s ≤2.OD nc ), moderate biofilm production (2.OD nc <OD s ≤4.OD nc ) and strong biofilm production (4.OD nc <OD s ) [10].

Tube method
A qualitative assessment of biofilm formation was done as described by Christensen et al. 1985 [9].
The TSB glucose (10 mL) was inoculated with a loop full of MRSA from overnight culture plates and incubated for 24 hours at 37°C. The tubes were decanted and washed with PBS (pH-7.2) and dried.
Then the tubes were stained by 0.1% crystal violet. Stain was removed by deionized water. Tubes were then dried in inverted position for biofilm formation. Biofilm formation was considered positive when a visible film lined the wall and bottom of the tube. Ring formation at the liquid interface was not indicative of biofilm formation. Experiments was performed in triplicate and repeated for three times.

Congo red agar method (CRA)
The congo red agar method was performed according to Freeman et al. 1989 [11]. The MRSA culture was streaked on surface of congo red agar and incubated at 37ºC for 24-48 hours. Black coloured colonies with dry crystalline consistency interpreted as positive biofilm producing strains. Red coloured colonies -interpreted as negative for biofilm production.

Determination of Minimum Inhibitory Concentration
The minimum inhibitory concentration of vancomycin (HiMedia, Mumbai, India) to MRSA isolates were screened by Microdilution method as suggested by CLSI 2012 [8]. The vancomycin powder was accurately weighed and stock solution of 128µg/ml was prepared. The known volume of 0.5 McFarland suspension of bacterial culture was added in each well containing the trypticase soya broth (TSB).
From the stock solution the different concentrations of drug ranging from 64µg/ml to 0.125µg/ml was made in in round bottom microtitre plates by serial dilution. The wells for positive and negative controls were even maintained in the plates. The microtitre plates were incubated at 37°C for 24 hours. The well with concentration of drug in which the growth of bacteria was inhibited was known to be the MIC.

Data analysis
The information collected from schedule was documented and tabulated. The data were statistically analyzed at 5% level of significance by SPSS version 16. A p-value of less than or equal to 0.05 was considered to be statistically significant (p≤0.05).

Gender-wise distribution of MRSA in sample population
Out of 400 samples taken, 123 were MRSA positive isolates out of which 67 (54.4%) were from males and 56 (45.5%) were from females.

Comparative study of MRSA isolated from total samples
In this study, all samples were analyzed for MRSA. Higher number of MRSA was isolated from hand (36.5%). The prevalence of MRSA isolates from nasal was 25% and from hand was 36.5%.

Comparative study of MRSA isolated from different samples
The MRSA isolated from hand were 73 and from nasal were 50. In 24 individuals the MRSA was isolated from both the hand and nasal. The higher frequency of MRSA was isolated from hand than in nasal. Maximum MRSA isolates were obtained from skin surface of barber's hand.

Comparative study of biofilm formation by MRSA in potential risk population
The biofilm formation assay showed that maximum isolates were biofilm producer. The biofilm producing MRSA was found maximum in beautician (56.2%), followed by barber (52.9%). Similarly, in municipal waste workers and healthy population biofilm producing MRSA were 48.2% and 46.4% respectively.

Biofilm formation assay
The biofilm forming ability of isolated MRSA was performed by three methods: microtitre plate method, tube method and congo red agar method.

Sensitivity and specificity of biofilm screening methods
The microtitre plate method was found to be most efficient standard method for studying biofilm formation as compared to tube method and congo red agar method. The parameters like sensitivity, specificity, negative predictive value, positive predictive value and accuracy were calculated. True positives were biofilm producers by microtitre, tube and congo red agar method. False positive were biofilm producers by tube method (TM) and congo red agar (CRA) method and not by microtitre method. False negative were the isolates which were non-biofilm producers by microtitre plate and CRA but were biofilm producer by microtitre method. True negatives are those which were non biofilm producers by all three methods [12].

Resistance pattern of biofilm producing MRSA
The biofilm producing MRSA showed resistance to cefotaxime, chloramphenicol, teicoplanin, cotrimoxazole, erythromycin, norfloxacin, trimethoprim. The non-biofilm producing MRSA showed resistance to tetracycline, ofloxacin, gentamicin, and ciprofloxacin. Ampicillin and amoxicillin were resisted by both biofilm producers and non-biofilm producers.

Multidrug resistant (MDR) CA-MRSA
Multidrug resistant community-acquired methicillin resistant S. aureus (CA-MRSA) were identified by their antibiotic resistivity pattern on, three or more than three commonly prescribed antibiotics of different classes. The prevalence of MDR CA-MRSA was 91%.

MIC of vancomycin to MRSA isolates from different gender population
The highest prevalence of VISA and VRSA were found to be in male population than in female population. However, the sample populations of female were less than that of male in our study.

MIC of vancomycin to MRSA isolates from hand and nasal sample
In this study, the highest prevalence of VISA was found to be in hand (31.7%) than in nasal (17.8%).
Similarly, the highest prevalence of VRSA was found to be in hand (21.9%) than in nasal (18.6%).

Discussion
Staphylococcus aureus is one of the common pathogens isolated in most microbiological laboratories [13]. It is responsible for a wide range of infections including superficial skin infections, food poisoning, osteomyelitis and septicemia [14].
Methicillin-resistant strains of staphylococci emerged by late 1970s and now have stood as prevalent as nosocomial pathogens [15]. The emergence of vancomycin resistant S. aureus has raised the concern that none of the antimicrobial drugs will be left to treat patients infected by these strains of staphylococci [16].
Our study was carried out at microbiology laboratory of  [23]. Around 73.1% of CA-MRSA isolates were resistant to erythromycin in our study. Our result supports the previous studies conducted in Nepal which reported increasing resistance of bacteria towards erythromycin and co-trimoxazole due to excessive use of their drugs to treat infections [21]. Even in our study the isolated superbug showed reduced sensitivity to erythromycin.
The susceptibility of ciprofloxacin was 88% observed in study conducted by Khatri et al. 2017 [24]. In our study the susceptibility to ciprofloxacin was 93.4% which was found high in comparison to other studies. Isolates MRSA were most sensitive to ciprofloxacin drug which was statistically significant with p<0.05. In study conducted by Mandelia et al. (2012) the susceptibility to ciprofloxacin was only 18.3% [23]. Chloramphenicol, co-trimoxazole and ciprofloxacin were much sensitive drugs in our study with statistical significance (p<0.05). Khatri et al. (2017) reported that higher MRSA sensitive to vancomycin followed by co-trimoxazole (84.2%) [24].
In Nepal, due to unnecessary use of antibiotic without doctor's prescription, the emergence of antibiotic resistance has been increasing. People can purchase antibiotic directly from pharmaceuticals, which has led to antibiotic resistant strains of microorganisms. However, the emergence of antibiotic resistant bacteria continues to threaten the ability to treat infections.
Recently, antibiotic resistant pathogens have been emerging in community, which may increase the impact they have on populations.
Antimicrobial resistance is an innate feature of bacterial biofilms and biofilm formation is higher in MRSA [25]. The biofilm forming ability of isolated MRSA was performed by microtitre plate method, tube method and congo red agar method. screening biofilm as compared to tube method and congo red agar method [12]. In our study, the microtitre plate method was found to be most sensitive and efficient method for quantitative screening of biofilm as compared to tube and congo red agar method. The screening of biofilm by microtitre method, tube method and congo red agar method was statistically significant (p<0.05).
The study conducted by Rezaei et al. (2013) reported that 15.4% of CA-MRSA was strong biofilm producer and rest 19.2% and 65.4% were medium and weak biofilm producer respectively [26].
The biofilm producing MRSA showed high resistance to cefotaxime, chloramphenicol, teicoplanin, cotrimoxazole, erythromycin, norfloxacin, trimethoprim. The non-biofilm producing MRSA showed resistance to tetracycline, ofloxacin, gentamicin, and ciprofloxacin. The resistivity pattern of biofilm producer and non-biofilm producer was statistically significant (p<0.05). Ampicillin and amoxicillin were resisted by both biofilm producers and non-biofilm producers. The resistant pattern of biofilm producing reported the ability of biofilm formation in drug resistance. However, non-biofilm producing antibiotic resistant was even found in our study. It can be explained that other than biofilm formation many other contributory role of bacteria is responsible for drug resistance.
Ciprofloxacin is known to be most effective against biofilm forming bacteria. In our study ciprofloxacin showed effectiveness to both biofilm producing and non-producing MRSA strains. In our study, only  [29]. The study indicates that biofilm producing ability might be one of the crucial factors for resistance towards the antibiotics.
In our study, 91% of CA-MRSA isolates were known to be MDR. Multidrug resistant bacteria were higher among biofilm producers which was also statistically significant (p<0.05).  [32]. Although vancomycin has been used as a drug of choice for treating MRSA, recently the VISA has been reported in CA-MRSA clones [33].
Detection of vancomycin resistance is essential not only for an optimal therapy, but also for infection control measures and epidemiological purposes. As recommended by the Clinical & Laboratory Standards Institute (CLSI), the broth dilution method was used for MIC determination for the detection of VISA and VRSA strains in this study. Most of the microbiological laboratories in Nepal depend upon disk diffusion method to identify susceptibility of S. aureus to vancomycin, which however cannot differentiate VISA and VRSA strains because of similar zone of inhibition. Detection of VRSA raises the concern and issue not only in clinical therapy but even in epidemiological settings.

Conclusions
In our study significant rise of VISA and VRSA were reported from CA-MRSA. The pathogen is spreading in community, hospitals and in wide areas. It can be warned that lack of hygiene, exposure Vancomycin sensitivity of total MRSA isolates