Site selection
MRSA samples were collected from three districts of Karnataka—Bidar, Kalaburagi, and Yadgiri, between April 2022 and September 2023. These sites were selected based on their healthcare density and waste-generation potential.

Geographic Information System (GIS) mapping
The spatial distribution of MRSA across the selected regions was analyzed using the Earth Explorer database (https://earthexplorer.usgs.gov/). The final three-dimensional vector maps were generated using ArcGIS 10.3 software. Sample coordinates were manually uploaded to the system to visualize the spatial distribution and determine the microbial diversity and severity across regions.²²

Sample collection
Biomedical waste samples were collected using appropriate personal protective equipment, including gloves, lab coat, and face masks, to ensure safety and prevent contamination. The waste was segregated on-site into specific categories: sharps, infectious waste, pathological waste, pharmaceutical waste, and non-hazardous waste, using color-coded containers in accordance with biomedical waste management guidelines. Red bags were designated for contaminated materials, such as swabs, needles, syringes, scalpel blades, and items saturated with blood or other potentially infectious fluids. Yellow bags are used for chemical, pharmaceutical, or cytotoxic residues, such as medication vials, intravenous tubing, or chemotherapy waste. Black bags were used for non-infectious materials, such as food waste and paper products, as shown in Figure 1. Swabs were collected from each waste category and submerged in separate flasks containing Luria-Bertani (LB) broth. The inoculated broths were incubated at 37 °C for 24 hrs to allow bacterial proliferation before further analysis.²³

Isolation of bacterial samples using serial dilution method
To isolate bacterial colonies, LB broth cultures containing biomedical waste samples were serially diluted. A 5 mL aliquot of the broth was transferred into a series of eight sterile test tubes, followed by serial dilution using 500 µL transfers at each step to achieve decreasing bacterial concentrations. Each dilution was thoroughly mixed to ensure uniform distribution. This method allowed for the quantitative estimation of the bacterial load and facilitated the isolation of distinct colonies upon plating. Nutrient agar media were prepared, poured into sterile Petri dishes under aseptic conditions, and allowed to solidify. Using a sterile inoculation loop, bacterial samples were collected from the diluted broth, and streak plating was performed in a zigzag pattern to ensure even distribution and isolation of individual colonies. The plates were incubated in an inverted position at 37 °C for 24 hrs to promote bacterial growth. Visible colonies were observed and sub-cultured for further studies.²⁴

S. aureus Screening on Mannitol Salt Agar (MSA)
Distinct colonies were selected based on morphological characteristics such as size, shape, and pigmentation. Selected colonies were screened on MSA (HiMedia, India) by spot inoculation and incubated at 37 °C for 24-48 hrs. Colonies exhibiting yellow coloration with surrounding yellow zones were considered presumptive S. aureus.²⁵

Microbial morphology profiling
Gram staining was performed to differentiate bacterial isolates based on their cell wall characteristics. Bacterial colony smears were prepared on clean glass slides and heat-fixed. Slides were sequentially stained with crystal violet (primary stain), iodine solution (mordant), ethanol (decolorizer), and safranin (counterstain). After air-drying, the slides were examined under a compound microscope at 10× and 40× magnifications to determine Gram staining. Gram-positive bacteria appeared purple, whereas Gram-negative bacteria appeared pink.²⁶

Biochemical characterization
Methyl Red (MR) test
MR-VP broth (HiMedia, India) was prepared as per manufacturer’s instructions, dispensed in 6 mL aliquots into sterile test tubes, and autoclaved at 121 °C for 15 min. Each tube was inoculated with two loops of test culture and incubated at 37 °C for 48 hrs. After incubation, 5-6 drops of methyl red indicator (pH range 4.4-6.2) were added. The development of a stable bright red color indicated a positive MR test result, confirming mixed-acid fermentation. Yellow color was recorded as negative.²⁷

Voges-Proskauer (VP) test
The inoculated MR-VP broth cultures were incubated at 37 °C for 48 hrs. To 1 mL of culture, 0.6 mL of Barritt’s reagent A (5% ±-naphthol in absolute ethanol) and 0.2 mL of reagent B (40% potassium hydroxide) were added sequentially. The tubes were shaken gently and allowed to stand at room temperature for 10-15 minutes. The appearance of a pink to red color indicated acetoin production (positive VP test).²⁸

Starch hydrolysis test
Starch agar plates were prepared using 1% (w/v) soluble starch (HiMedia) in a Nutrient Agar base. After autoclaving and solidification, the test strains were streaked linearly and incubated at 37 °C for 72 hrs. The plates were flooded with Gram’s iodine solution and observed after 5-10 min. A clear halo around the growth indicated starch hydrolysis (amylase activity), whereas a dark blue color indicated no hydrolysis.²⁹

Hemolytic activity assessment
Hemolytic activity was quantified on 5% defibrinated sheep blood agar plates prepared and poured under sterile conditions: isolates were stab-inoculated or streaked in a single line and incubated at 37 °C for 18-24 hrs in ambient air (or 5% CO₂ for fastidious organisms). Plates were visually inspected against a black background for areas surrounding colonies: β-hemolysis was noted as a clear, translucent area of total red blood cell destruction; α-hemolysis as a greenish-brown hemolysis or met-hemoglobinization of agar below and around colonies; and γ-hemolysis as no visible change. Where hemolysis was weak or dubious, plates were re-examined at 24-48 hrs and incubated under other atmospheric conditions if clinically warranted; photographic records and measurement of hemolytic zone diameter were conducted for comparative studies, and sterility and media controls were added to validate blood agar quality.³⁰

Gram staining
Gram staining is a microbiological method used to distinguish various types of bacterial cells based on their cell wall composition. A small sample was taken from each of the sub-cultured colonies of randomly collected from three different regions: samples 1, 2, and 3. The samples were spread onto separate slides and heat-fixed to allow adherence to the bacterial cells. The slides were then flooded with crystal violet stain and left to sit for the designated time. The excess stain was then rinsed with water. An iodine solution was applied as a mordant to enhance staining. The slides were then briefly treated with a decolorizing agent such as ethanol or acetone to differentiate between Gram-positive and Gram-negative bacteria. The slides were then rinsed again with water. A counterstain, typically safranin, was applied to color the Gram-negative bacteria. The slides were then rinsed once with water, excess moisture was blotted off, and the slides were allowed to air dry. The stained slides were observed under a microscope at 10× and 40× magnificent to determine the Gram staining of the bacteria.³¹

Molecular characterization
The bacterial culture was resuspended in 1 mL of DNAiso Reagent (Takara Bio) and mixed thoroughly by pipetting to ensure complete homogenization. The samples were incubated at room temperature for 5 min and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a fresh tube, and DNA precipitation was performed by adding absolute ethanol (0.5 mL), followed by gentle inversion. After incubation for 3 min at room temperature, the mixture was centrifuged at 10,000 rpm for 10 min to obtain a DNA pellet. The pellet was washed twice with 75% ethanol, air-dried for 20-30 min, and dissolved in 30 µL Milli-Q water. The quality of the extracted DNA was assessed using 1% agarose gel electrophoresis, and the DNA was stored at -20 °C until further use. PCR amplification of the 16S rRNA gene was performed in a total reaction volume of 20 µL, containing 1 µL of bacterial DNA (10-50 ng), 1 µL each of forward and reverse primers, 10 µL EmeraldAmp GT PCR Master Mix (Takara Bio), and nuclease-free water to make up the volume. The universal primers 27F (5′ -AGAGTTTGATC(AC)TGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used for amplification. The PCR cycling conditions consisted of initial denaturation at 94 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, extension at 72 °C for 1.5 min, and a final extension at 72 °C for 10 min. The amplified PCR products were resolved on a 1% agarose gel containing ethidium bromide in 1× TAE buffer at 120 V for 45 min, along with a 100 bp DNA ladder, to confirm amplification. The PCR products were sequenced using the BDT v3.1 Cycle Sequencing Kit on an ABI 3730xl Genetic Analyzer. The forward and reverse sequences obtained were assembled to generate a consensus sequence using the Aligner software. The consensus sequence was subjected to BLAST analysis against the NCBI GenBank database (“nr” database). Based on the maximum identity scores, closely related sequences were selected and aligned using ClustalW in MEGA 11, and a phylogenetic tree was constructed using the Neighbor-Joining method to determine the taxonomic position of the isolate.³²

Virulent factor characterization
Nitrate reductase test
Reduction of nitrate was determined in nitrate broth with 0.1-0.2% KNO₃: the culture was inoculated into 5-10 mL of sterile nitrate broth and incubated aerobically for 24-48 hrs at 37 °C (extended incubation up to 72 hrs may be applied for slow reducers), using uninoculated broth as the negative control and a positive control being a known nitrate-reducing organism. Following incubation, 0.5 mL quantities were taken into sterile tubes (or directly added reagents to culture) and sequentially added reagents A (sulfanilic acid) and B (α-naphthylamine) (usually 0.5 mL each); red/pink color development immediately is a sign of nitrite production (nitrate → nitrite). If no color is seen, a pinch (10 mg) of zinc dust is added: appearance of red after zinc suggests presence of unreduced nitrate (i.e., organism did not reduce nitrate), while the absence of any color following addition of zinc suggests complete reduction of nitrate to beyond nitrite (to N₂ or other nitrogenous gases), taken as a positive reduction; results are noted and matched with gas production (where Durham tubes have been used), incubation period, and repeat testing if required for weak reactions.³⁰

Antimicrobial Activity-Drug Resistance Test (Agar Disc Diffusion/Kirby-Bauer)
Antimicrobial susceptibility was conducted by the standardized Kirby-Bauer disc diffusion technique on Mueller-Hinton agar (MHA) following CLSI recommendations: log phase cultures of bacteria were inoculated to a 0.5 McFarland turbidity standard (~1.5 × 10⁸ CFU/mL) in sterile saline; within 15 min of inoculation, a sterile cotton swab was wetted with the suspension, excess liquid removed by pressing against the tube wall, and the entire MHA plate surface inoculated by streaking in three directions to achieve an even lawn and permit the plate surface to dry (5-10 min) prior to application of antibiotic discs. Commercial antibiotic discs (lot and manufacturer documented) were gently applied on the inoculated agar using sterile forceps or an automatic disc dispenser with at least 24 mm center-to-center spacing; plates were inverted and incubated for 18-24 hrs at 37 °C. Following incubation, zone diameters of inhibition were measured to the nearest millimeter with a caliper or ruler, tabulated in mm, and interpreted according to the most current CLSI breakpoints as Susceptible, Intermediate, or Resistant. Quality control organisms were run concurrently to confirm the procedure, and all testing was repeated for any questionable zones or confluent growth on plates or other technical problems.³³

Computational analysis
PICRUSt2: Functional profiling
Functional profiling of the identified bacterial isolate was performed using PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States). The 16S rRNA gene sequence, along with the corresponding OTU table, was used as the input for the PICRUSt2 full pipeline executed on the Galaxy Europe platform. The workflow involved phylogenetic placement of the input sequence into a reference tree, followed by the prediction of gene family abundance using evolutionary modeling. Based on this, functional annotations were assigned in terms of KEGG Orthologs (KO) and Enzyme Commission (EC) numbers, which were further mapped to metabolic pathways. The Nearest Sequenced Taxon Index (NSTI) value was used to assess the reliability of the predictions, where lower NSTI values indicated closer similarity to reference genomes and higher confidence in the inferred functions. Predicted pathway abundances were used to evaluate the metabolic and functional potential of the isolates. Notably, PICRUSt2 provides predictive functional profiling based on 16S rRNA data, and does not represent the presence or expression of experimentally validated genes.³⁴