Bacterial strains were isolated from environmental samples collected from within the campus and kept as stocks for routine culturing in the general microbiology laboratory in the Department of Applied Biology, University of Sharjah; these are: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Streptococcus pyogenes. For long-term storage, the strains were preserved in 50% glycerol stocks at −80˚C and for experimentation, strains were grown overnight on nutrient agar at 37˚C (Nutrient agar (Himedia Laboratories, Maharashtra, India, Cat. No. M001).

Both antibiotic activity and the multi-resistance profile were conducted for the tested strains using the inhibition zone method and the VITEK-2 system and the automated VITEK® SOLUTIONS—Complete automated ID/AST platform ( https://www.biomerieux-diagnostics.com ). The multi-resistance of the tested strains was determined manually by dividing the number of the resisted antibiotics on the total number of the tested ones; as this is according to the international standards as stipulated in the Clinical and Laboratory Standards Institute (CLSI) CLSI M100-Ed35, 2025 [24] . Four multi-resistance control strains were used for quality control assurance; these were Escherichia coli, ATCC® 25922, Klebsiella pneumoniae, ATCC® 13883, and Streptococcus faecalis, ATCC® 33186, and were subjected to the same diagnosis protocol as with the tested strains.

The bacterial strains were grown overnight on nutrient agar plates at 37˚C. No less than four colonies were collected by using a sterile loop and resuspended in a 2 mL saline solution and then vortexed to obtain a homogenous mixture. The suspension was adjusted to the 0.5 McFarland standard (~1.5 × 108 CFU/ml) and tested for antibacterial activity using well diffusion.

Antibiotic sensitivity tests were performed using the Kirby-Bauer disk diffusion assay [25] . The bacterial mixtures were seeded on Mueller-Hinton agar plates by using a sterile cotton swab. Antibiotic discs were then placed on the agar plates using sterile tweezers. The plates were incubated overnight at 37˚C. After 24 hours, the inhibition zones were measured using a ruler, and the data were interpreted as sensitive, intermediate, and resistant according to the Clinical and Laboratory Standard Institute and compared also with the manufacturer reference sheet. Additionally, VITEK antibiotic susceptibility testing analysis was conducted by growing the bacterial strains overnight on nutrient agar at 37˚C. Then, using a sterile loop, four to five colonies were collected and resuspended in a 2 mL saline solution. The density of the colonies was checked using a Densi CHEK Plus device; the reading should range from 0.58 to 0.62. The mixture was put on the cassette, then VITEK® 2 Antibiotic Susceptibility Testing Gram-Negative (AST-GN70) cards and VITEK® 2 Antibiotic Susceptibility Testing Gram-Positive (AST-GP75) cards were placed accordingly in the VITEK® 2 Compact diagnostic system (bioMérieux https://www.biomerieux.com/ ).

The Minimum Inhibition Concentration (MIC) and biochemical characteristics were conducted using the VITEK® SOLUTIONS—Complete automated ID/AST platform ( https://www.biomerieux-diagnostics.com ). Bacterial strains were grown overnight on nutrient agar at 37˚C by using a sterile loop; four to five colonies were collected and resuspended in 2 mL saline solution. Density of the colonies was checked using a DensiCHEK plus device; the reading should range from 0.58 to 0.62. The mixture was put on the cassette, and VITEK 2 Identification Card Gram-Negative (ID GN) cards and VITEK 2 Identification Card Gram-Positive (ID GP) cards were placed accordingly. Samples were placed in the VITEK 2 Compact device.

Genomic DNA was extracted from 1 ml of pure bacterial culture using the Isolate II Genomic DNA kit (Bioline, BIO-52066) according to the manufacturer’s protocol. The purity and concentration of extracted genomic DNA were assessed using the NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific) ( Table 1 ). To amplify the 16S rRNA gene, the MicroSEQ Full Gene 16S rDNA PCR Kit (Thermo Fisher Scientific, 4349155) was used. In brief, 60 ng of genomic DNA was used in three separate PCR reactions, thereby amplifying three overlapping fragments to ensure that a high-quality and full-length gene sequence was obtained. PCR was carried out according to the following conditions: 95˚C for 10 minutes; 30 cycles of 95˚C for 30 seconds, 60˚C for 30 seconds, and 72˚C for 45 seconds, followed by a final extension at 72˚C for 10 minutes. Generated amplicons were then verified by visualizing them on a 2% agarose gel. Moreover, 5 µL of each PCR product was treated with ExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher Scientific, 78200.200.UL) according to the supplied protocol and used for sequencing. For contamination checks during DNA extraction, PCR, or sequencing, negative and positive control as provided by the manfcturer within the kit were run in parallel with samples.

Sequencing reactions were conducted using the MicroSEQ Full Gene 16S rDNA Sequencing Kit (Thermo Fisher Scientific, 4347484) which includes the primers in its master mix solution according to the manufacturer’s protocol. In short, each of the three purified PCR products was sequenced using both their respective forward and reverse primers, resulting in six reactions. Sequencing reactions were carried out using the following conditions: 25 cycles of 96˚C for 10 seconds, 50˚C for 5 seconds, and 60˚C for 4 minutes. Sequencing reactions were then purified using the ethanol/EDTA/sodium acetate precipitation method. Afterward, capillary sequencing was carried out by the Genetic Analyzer 3500 (Applied Biosystems, Thermo Fisher Scientific, USA). Obtained forward and reverse sequences were then analyzed using the Sequencing Analysis Software 6 (Applied Biosystems, Thermo Fisher Scientific, USA) and assembled into one contiguous sequence using the CAP3 software ( http://doua.prabi.fr/software/cap3 ). Finally, the assembled sequence was aligned to published sequences using Basic Local Alignment Search Tool (BLAST) ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ).

The full gene sequence of the bacterial strain extracted was aligned automatically using the BLAST tool against the gene libraries available for bacterial species in the NCBI ( https://www.ncbi.nlm.nih.gov/ ), Sanger Institute ( http://www.sanger.ac.uk ), DDBJ ( http://www.ddbj.nig.ac.jp ), and EMBL-EBI GeneBank ( http://www.ebi.ac.uk ) databases.

The phylogenetic analysis was constructed using the Neighbor-Joining tree of the isolated strain using BLAST and CLUSTAL W (1.83) available in the following international GeneBanks: National Center for Biotechnology Information (NCBI), International Nucleotide Sequence Databases (INSD) (DDBJ GenBank), and EMBL-EBI Bank (European Bioinformatics Institute and the European Molecular Biology Laboratory). The closely related homologous strains were identified, retrieved, and compared to the sequence of the strain extracted using CLUSTAL W (version 3.2) available on the Biology StudyBench ( https://workbench.sdsc.edu/ ).

Genotyping identification of species through sequence similarity was determined based on the international diagnostic criteria as set by the international Gene Banks (International Nucleotide Sequence Databases (INSD) (DDBJ GenBank), EMBL-EBI Bank (European Bioinformatics Institute and the European Molecular Biology Laboratory), and the National Center for Biotechnology Information (NCBI), where if the difference between the query and the compared strain is 1 - 1.5% (14 - 22 bp), 1.5 - 5.0% (23 - 72 bp), and 5.0 - 7.0% (72 - 98 bp), then the query strain should be given to the same species, genus, or a different genus, respectively.

The biochemical characteristics diagnosis of the tested strains were tested against 64 biochemical reactions as included in the VITEK-2 system (data not shown). Several quality control strains were used for assurance and were subjected to all diagnosis protocols as with the tested strains, including Escherichia coli, ATCC® 25922, Klebsiella pneumonia, ATCC® 13883, and Streptococcus faecalis, ATCC® 33186.

The multi-resistance profile of strains revealed a high prevalence level of resistance as follows: E. coli 12.5% (E1), P. aeruginosa 37.5% (F1), P. aeruginosa 31.25% (H1), P. aeruginosa 31.25% (G1), P. aeruginosa 37.5% (B2), K. pneumoniae 6.25% (A1), K. pneumoniae 50% (C1), K. pneumoniae 6.25% (D1), K. pneumoniae 6.25% (D2), S. pyogenes 0% (L1), for the isolates E. coli isolate, P. aeruginosa isolates, K. pneumoniae isolates, S. pyogenes isolate, respectively ( Table 2 and Table 3 ). The most resisted antibiotics by the strains were ampicillin (Amp) 10 mcg, ciprofloxacin (CIP) 40 mcg, nitrofurantoin (NIT) 100 mcg, and co-trimoxazole (sulfa/trimethoprim) (COT) for the strains K. pneumoniae; Pseudomonas aeruginosa and 12.5% for E. coli respectively. For quality assurance, the ATCC strains, including Escherichia coli, ATCC® 25922, Klebsiella pneumoniae, ATCC® 13883, and Streptococcus faecalis, ATCC® 33186, were used as controls as subjected to the diagnosis protocol as with the tested isolates. The inhibition zones varied from 0 mm for the multi-resistance strains for as low as 10 mcg of ampicillin (Amp) to 40 mm for the sensitive strains as high as 40 mcg ciprofloxacin ( Table 2 and Table 3 ).

for E. coli. Our present results of the multi-antibiotic resistance profile agree with the latest reported data on the global surveillances by the WHO [26] . Furthermore, in comparison with the latest regional and global infections caused by the multi-resistance strains [27] [28] . Our results revealed high similarity among other bacteria strains which have been reported causing the most common infections: ear, nose, respiratory, and urinary tract infections (UTI), respectively.

The phylogenetic analysis of the top-5 species shows conclusively the accurate genotypic diagnosis for all strains with homology of 99.9% and above. This underlined the importance of using phylogenetic genotyping in the diagnosis of such pathogenic strains causing various infections reported worldwide [29] . The multi-resistance profiling order was ampicillin (AMP) 10 mcg, cefuroxime (CXM) 30 mcg, nitrofurantoin (NIT) 100 mcg, and co-trimoxazole (sulfa/trimethoprim) (COT) for the pathogenic strains K. pneumoniae and Pseudomonas aeruginosa, respectively.

The importance of these results in this study shows conclusively its valuable proven medical implications to enable clinicians to quickly optimize antimicrobial therapy and implement infection control policies, including the right medical decision for the right antibiotic in the right dose at the right time. Furthermore, it improves on the multi-resistance profiling on surveillance programs of the infectious diseases in the UAE and worldwide.