Characterization and Identification of Methicillin-resistant Staphylococcus aureus (MRSA) Producing Biofilm: Impacts of Garlic Extract and Lactobacillus Biosurfactants

1Department of Medical Microbiology, Faculty of Applied Medical Sciences, Taif University, Turrabah, 21995, Saudi Arabia. 2Department of Microbiology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt. 3Animal Reproduction Research Institute, Immunobiology and Immunopharmacology Unit, Giza, Egypt. 4Clinical Laboratory Sciences Department, Turabah University College, Taif University, Turabah, 29541, Saudi Arabia. 5Biochemistry Department, Faculty of Veterinary Medicine, Benha University, Benha 13736, Egypt. 6Chemistry Department, Turabah University College, Turabah 29541, Taif University, Saudi Arabia. 7Zoology and Nematology Department, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt *Corresponding Author E-mail : esam2005micro@gmail.com

Staphylococcus aureus (S. aureus) is a typical pathogen,causing a great variety of infections on skin 1 . Treatment of S. aureus infections with antibiotics is frequently less powerful to insufficient because of the development of antibiotic-resistance strains, Methicillin-resistant S. aureus (MRSA) 2 . The virulence of S. aureus pathogen relies upon the generation of several factors. There were 24anchored cell wall proteins that expressed by S. aureus. These proteins promote S.aureus to cling to extracellular materials, influencing on the invasion of non-phagocytic cells and interference with innate immunity 3 .
MRSA was isolated and known by followings, hospital related MRSA (HA-MRSA), community-related MRSA (CA-MRSA) and livestock-associated MRSA (LA-MRSA) 4 . Nowadays, MRSA strains were isolated from different foods sources, such as poultry, pork, beef, milk and vegetables, concluded that foods may act as source of contamination 5 . S.aureus biofilmis more significant virulence factor than any other strains. The development of biofilms on medical devices is a major issue in hospitals, as they can turn into a wellspring of disease 6 . S. aureus hinder the first line of defense of in the body known as immune system by building a wide variety of active peptides 7 .
Garlic (Allium sativum L.) contains active antibacterial substance called allicin, diallyl disulfide and alipin, which is powerful against numerous pathogenic microbes 8,9 .S-allyl-L-cysteine sulfoxide (alliin) is a garlic organosulfuring redient with no odor. The allinase enzyme, which is the cysteine sulfoxidelyase,turnsalliin into allicin when garlic is cut. The strong smell,antioxidant and antibacterial functions of garlic are attributed to allicin 10 . The main biological, biochemical and antioxidant active substance of fresh garlic is named allicin. Probiotic bacteria, for example, Lactobacillus fermentum and Lactobacillus plantarum, found to repress S. aureus biofilm formation, however the mechanism is not well known. S. aureus genes ica A and icaD expression is engaged with the synthesis of the biofilm matrix 11 .
The Current study was planned to isolate, characterize and identify local MRSA isolates with regards to genotypic and phenotypic features. Moreover, the activity of garlic water extract (gWE) and lactobacillus biosurfactants against biofilm formation by MRSA isolates were examined.

Specimen collection and identification
A total of 130 samples were isolated and collected from four sheep farms and local grocery stores in Turabah governorate, Taif, Saudi Arabia. The collected samples were divided as following, 90 out of 130 samples were nasal swabs, 10 were lung tissues from recently dead sheep suffering from respiratory symptoms and 30 were raw sheep meat collected from local grocery stores. Collection of samples were ranged from October 2017 till April 2018. All samples obtained from live and dead sheep were obtained by veterinarians the guide lines of Taif University, Saudi Arabia. The collected nasal swabs and lung tissue samples were sub-cultured on tryptic soy broth (Difco) and then incubated for 24 hrs at 37 °C and were maintained on trypticase soy agar, (Difco). The meat samples (raw sheep)were taken and kept at 4 °C prior to investigation. 25 grams of meat samples were homogenized with buffered peptone water (225 ml) and 1:10 serial dilution was done. 0.1 ml of diluted samples were streaked on Baired-Parker (BP) agar (Oxiod) supplemented with egg yolk tellurite emulsion(Oxiod).The plates were then incubated for 24 hrs at 37 °C 12 .Isolates were primarily identified by Gram stain, Catalase, Culturing on mannitol salt agar, coagulase test 13 andmolecular PCR technology.
After the confirmation of S. aureus from previous routine microbiological methods, isolates were identified for MRSA by cefoxitinand oxacillin disc diffusion test according to NCCLS guidelines,2002 14 . Next, All resistant isolates to oxacillin (1 μg) and cefoxitin (30 μg) discs were confirmed by PCR technology for MRSA.

DNA extraction of MRSA and Triplex PCR identification
A triplex PCR technology was used to identify and discriminate the S. aureus from other staphylococcal species and MRSA isolates genotypically 15 . Then 500 µl of the broth was centrifuged for 5 minutes at 2000 rpm. For the pellet, 500 μl of DEPC-treated water (DNase-RNase free) was added and flushed in a vortex.
After boiling in water bath at 100ºC for 10 minutes. The rubes were centrifuged at 10000 rpm for 5 min, the supernatant contain bacterial DNA was taken and used as a template for PCR. PCR (triplex)was used to identify S. aureus from other Staphylococci species and to characterize MRSA genotypically. To detect Staphylococcus spp.,16S rRNA gene was used. To detect S. aureus specific gene, nucgene was used. The mecA expression as a methicillin resistance specific gene was used. The sequence of sense and antisense primers for each gene was used ( Table  1).Exactly 5 ml of the rapid extracted DNA were taken as a template in a 25 μL PCR mixture. The PCR reaction was consisted of 5µL of cDNA, 2 µL of 10 pM of each primer, and 12.5 µL master mix for PCR (Promega Corp, Madison, WI, USA). The volume was adjusted to 25 µL with sterile deionized water. PCR reaction was performed in thermal cycler machine (Bio-Rad T100 TM ). The primers were synthesized from Macrogen Co, Seoul Korea. The DNA amplification was performed as follows: 94ºC for 4 minutes of initial denaturation; 30 cycles of 94ºC for 50seconds, 58ºC for 50 seconds and 72ºC for 90 seconds; and a final extension at 72ºC for 7 minutes. Amplified genes were loaded in 1.5% agarose gel (stained with ethidium bromide). The 756-bp (16S rRNA), 310-bp (mecA) and 279-bp (nuc) amplified DNA fragments were separated by electrophoresis in agarose gel and visualized under UV-light using gel documentation system.

Congo red Agar method for determination of biofilm production (CRA)
To detect biofilm production from MRSA isolates, a specific medium. The medium was composed of BHI; 37 gms/L, sucrose; 50 gms/L, agar agar; 10 gms/L, and congo red stain; 0.8 gms/L was used. Congo red medium was prepared and autoclaved. Then the MRSA isolates were inoculated in plates with the medium and incubated for 24 and 48 hrs at 37°C 16 .Black (intense black) were considered strong positive. While dark colonies (black colonies) considered intermediate biofilm producers. Pink colonies mean weak biofilm producers, while the non-biofilm producers strains remain red with smooth appearance. The experiment was carried out three times 17 .

Microtitre plate test (MTP) for quantification of slime production of MRSA
The quantification of biofilm formation of isolated MRSA and typical positive on CRA was done as described before 18 , with little modifications. Isolates of MRSA were subcultured on TSB supplemented with 0.25% glucose overnight at 37°C with shaking. About 100µl were added to microtitre plate (96-well) contains 100µl of fresh TSB supplemented with 0.25% glucose. The plate was incubated for 24hrs at 37°C. Then, plates carefully washed by PB(PH 7.4) three times to remove none adherent cell. Then plates were dried in the air and maintained in inverted position before staining. Crystal violet (0.4%) was used to stain the adherent cells with a volume of 100µl per well for 120 seconds and then left to be air-dried and washed by sterile distilled water three times. Finally,Optical density (OD) was measured using spectrophotometer (Smart Spec tm Plus-Bio-Rad-USA) at 570nm.The slime produced from isolates were classified into 4 categories based on obtained OD, strong slime formers (OD 570 ≥ 3.0), moderately slime formers (OD 570 ≥ 1.5-2.0), weak slime formers (OD 570 ≥ 0.5-1.0) and none slime formers were (OD 570 ≤ 0.5).

Preparation of garlic water extract (gWE)
Garlic was purchased from local markets in Turabah, Saudi Arabia. Buds were cleaned thoroughly, then rinsed with tap water and left for drying for 24hrs at room temperature. Fifty grams of garlic was crushed in a sterile grinder  to obtain fine powder. Then 100 ml of sterile pure water were added to the powder and was left for overnight, filtration was done using Whatman paper No1. Finally, the extract was concentrated using shaking incubator (Model: TH2-300-SN 170824647-China) at 37°C for 6days. The obtained concentrated garlic extract was kept in deep freezer till use 19 .

Isolation of Lactobacillus bio surfactants (LAB) from Yoghurt
LAB was isolated from yoghurt purchased from local markets in Turabah, KSA. Briefly;one gram of yoghurt and9 mL sterile saline water were mixed. Then serially diluted and immediately plated on LB agar(Difco, Bacton, USA), which is selective for Lactobacillus spp. The plates were kept and incubated anaerobically, at 37 °C for 48 hrs. Catalase-negative, gram-positive and rod-shaped bacilli were considered LAB positive, further confirmation was made by culturing of previous obtained colonies on MRSA agar 20 .

Anti-biofilm Activity
The anti-biofilm activity was explained before 21 with some modifications. All isolates of MRSA were sub-cultured overnight with fresh sterile tryptic soy broth (TSB) supplemented  6 ) were 87.5%, 75% and 5% in nasal , lung and raw meat respectively with 0.25% (w/v) glucose. Then, 100 µl of cultures of each isolate was transferred to 96-well microtitre plates. A volume of 100 μL of different concentration of garlic extract (12.5, 25, 50 and 100mg/ml) and LAB biosurfactants(50 and100mg/ ml) were added to each well except positive and negative control wells. Wells were incubated at 37 °C for 48 hrs, then gently washed twice with sterile PBS (PH 7.4) 22 . Thereafter, the biofilm were fixed using 200 µL methanol for 10 min, then stained with 0.1% crystal violet for about 10 min and rinsed 3times with distilled water. The adherent cells were dissolved using 200 μL of 33% acetic acid, OD was measured using spectrophotometer (Smart Spec tm Plus-Bio-Rad-USA) at 570nm 20 . Each experiment was repeated three times. Positive control contains bacterial isolates without any treatment but negative controls contains only tryptic soy broth with 0.5% (w/v) glucose. The current results were expressed in biomass formation inhibition percentage 23 .

Statistical analysis
Data are calculated and expressed as means ± standard error for values of MRSA isolates. All data analyzed using ANOVA (analysis of variance) by Bonferroni test for SPSS software version 11.5 for Windows (SPSS, IBM, Chicago, IL, USA). All values less than P < 0.05 wee considered statistically significant.

RESULTS
Isolated S. aureus from nasal swabs, lung tissues of dead sheep and raw sheep meat were tested using agar disc diffusion method to examine the MRSA distribution ( Figure 1). The obtained results revealed that 15 isolates (8, nasal; 4, lung and 3, raw sheep meat) were positive S.aureus MRSA specific species.
Triplex PCR was used to discriminate S. aureus from other S. species. Triplex PCR targeted the 16S rRNA, the nuc and the mec Agenes to be specific genes for Staphylococci, S. aureus and S. aureus specific MRSA, respectively. Detected DNA fragments were with size of 756, 279 and 310 bp, for the 16S rRNA, the mecA and the nucgenes, CRA test was used for biofilm detection, black colonies with slimy texture revealed positive biofilm MRSA producers, while red colonies indicated none biofilm producing strains ( Figure  3A-B).All S. aureus MRSA were cultured on CRA to differentiate between biofilm and none biofilm producers. The obtained results denoted that 12 out of 15 examined MRSA isolates (80%) were considered biofilm producers (black color) meanwhile three isolates (20%) were red color that means negative biofilm producers (Figures3  and 4). MTP test confirmed the results obtained by CRA. The obtained data was the same as that of CRA as seen in Figure 5.
S. aureus MRSA biofilms were exposed to garlic extract (gWE). The results of gWE reported that S. aureus MRSA biofilm was inhibited significantly after initial incubation with 25 mg/ml garlic extract (Fig. 5). In parallel, Figure6 showed the inhibitory effect of Lactobacilli biosurfactants on biofilm generation by MRSA. Compared to control,S. aureus MRSA biofilm was repressed by Lactobacilli biosurfactants at 50 and 100 mg/ml for 18 hrs.

DISCUSSION
MRSA has gained increasing importance in veterinary medicine in the past 2 decades, as MRSA shows resistance not only b-lactams but also other classes of antimicrobials 24,25 .The distribution of MRSA and its several antibiotics resistance are growing worldwide 26 .The prevalence of MRSA in large ruminant farms was 34% in milk samples collected from Pakistan 27 . The presence of MRSA in samples of meat was previously confirmed [28][29][30] , while it varies greatly by geographical location. Lower results from retail meat (˂ 5%) were reported before 31,32 , while Similar results (10-12 %) were shown in other studies 33,34 . Other, confirmed that MRSA prevalence was 31.8% in cattle, 29.8% of sheep, and 11.5%in goats in Jordan 35 .
Our findings showed that MRSA isolates were detected in 20% of our samples and the difference from other results may be attributed to the origin of samples used as they used milk samples while ours are nasal, lung and raw meat samples. The mecA gene detection by triplex PCR technology is a gold tool to detect MRSA strains compared to conventional disc diffusiontest 36 .
During the past two decades, scientists confirmed the increase in prevalence of mecA gene with multiple antibiotics resistance 37 .
Nearly, all mecA positive isolated S. aureus strains exhibited resistance to oxacillin and cefoxitin 38 . These results go ahead with our reported results, where, all isolated MRSA with mecA gene were all resistant to oxacillin and cefoxitin using disc diffusion test. The nuc gene was found also in all isolated MRSA strains, as it targeted S. aureus specific isolates and was more species specific than 16S rRNA(S. species specific) 39 . The high percent reported may be attributed to the high use of antibiotics as growth promoters in animals. Same was confirmed in a previous reported study in Jordan (29.8%) 35 .
The biofilms helps the bacterium to resist death and still survive within the host, then cause chronic or persistent infections 40 . Biofilm formation plays a vital role in pathogenicity, virulence and antimicrobial resistance to antibiosis 41 .The ability of pathogenic bacteria, such as S. aureus, to produce biofilm is the cause for persistent infections 42 . Bacteria producing biofilm are resistant to antibiotics, compared with non-biofilm producers 43 . The degree of biofilm formation by MRSA depends on the intercellular adhesion molecules production, named intercellular adhesion (ica) loci that includeica A, icaB, icaC, and icaD genes 44 .The process of biofilm formation consists of two independent processes: bacteria initially, attached to a solid surface. followed by proliferation and accumulation of bacteria cells, and thus resulted in biofilm formation and maturation 45 .
There are different methods for phenotypic detection of slime production as CRA which used in our study and considered rapid reliable qualitative test. CRA contains congo red dye that can detect the polysaccharides on the cell wall of both Gram positive and Gram negative bacteria 16 .The obtained results showed that(12/15)of MRSA isolates were considered as biofilm producers (black color) (80%).CRA was used in Previous studies and the biofilm producing MRSA( black color)was confirmed and was with a percentage of 85.1% 46 .MTPis another phenotypic test but more convenient and quantitative than CRA depending on measuring of OD using spectrophotometer and can detect S. aureus cell wall polysaccharides adhering to the polystyrene plate directly. The obtained results are coincided with that of CRA. Previous studies showed that (93/94) MRSA isolates were biofilm producers using the microtitre plate test 47 .
The anti-biofilm impact of water extract garlic (gWE) was investigated. A reduction in biofilm generation was reported at a concentration equivalent to 0.25 mg/ml gWE. The effect of gWE can be attributed to its active components that stifled the biofilm formation 48 .In parallel, previous reports confirmed that bio-surfactants separated from Lactobacilli strains has anti-biofilmactivity against MRSA. This findings that potentiate our results increase the usage of these natural products as an alternate promising therapies for treatment of S. aureus MRSA strains 49 .

CONCLUSIONS
Current study characterized and determined the prevalence of S. aureus MRSA in Turabah, Saudi Arabia using well established microbiological and molecular techniques. Moreover, confirmed the anti-biofilm activity of garlic and lactobacillus biosurfactants as promising medications for treatment of S. aureus MRSA in animals.