* To whom all correspondence should be addressed. Tel.: 00966530741541, Fax: 0096627274299; E-mail: khyate_99@yahoo.com JOURNAL OF PURE AND APPLIED MICROBIOLOGY, June 2018. Vol. 12(2), p. 489-496 Molecular Identification and Phylogenetic Analysis of Multidrug-resistant Bacteria using 16S rDNA Sequencing Walaa F Alsanie 1 , Ebaa M Felemban 2 , Mona A. Farid 3 Mohamed M Hassan 4,5 , Ayman Sabry 4,6 and Ahmed Gaber 4,7 * 1 Department of Clinical Laboratories, Faculty of Applied Medical Sciences, Taif University, Saudi Arabia. 2 Department of Nursing, Faculty of Applied Medical Sciences, Taif University, Saudi Arabia. 3 Genetics Department, Faculty of Agriculture, Kafrelsheikh Univ., 33516, Kafr El-Sheikh, Egypt 4 Department of Biology, Faculty of Science, Taif University, Saudi Arabia. 5 Department of Genetics, Faculty of Agriculture, Minufiya University, Egypt. 6 Department of Cell Biology, National Research Center, Dokki, Giza, Egypt. 7 Department of Genetics, Faculty of Agriculture, Cairo University, Egypt. http://dx.doi.org/10.22207/JPAM.12.2.07 (Received: 20 March 2018; accepted: 12 May 2018) In the present study, 30 multidrug-resistant bacterial samples were isolated from different hospitals in the Taif governorate in Saudi Arabia. Given its discriminating power as a universal phylogenetic marker, the 16S rDNA gene was sequenced in a comprehensive diversity study to determine the evolutionary and phylogenetic relationships among the bacterial isolates. The 16S rDNA genes of all isolates were successfully amplified using PCR, and comprehensive identification results were based on GenBank databases. Analysis revealed nucleotide identities ranging from 76% to 100% based on the consensus sequences of 21 species, namely, Bacillus cereus, Bacillus subtilis, Bacillus tequilensis, Caldimonas manganoxidans, Citrobacter freundii, Enterococcus faecium, Escherichia fergusonii, Klebsiella pneumoniae, Lactobacillus plantarum, Lactococcus garvieae, Leuconostoc mesenteroides, Myristica yunnanensis, Pantoea eucrina, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus petrasii, and Staphylococcus saccharolyticus. We observed high variability in terms of DNA length and GC content between and within species. Phylogenetic analysis clustered the isolates into three groups. The number of sites ranged from 827 (S. aureus) to 1,219 (L. mesenteroides). Estimation of nucleotide diversity (ð) showed that all analyzed sequences were diverse site- wise and also exhibited high nucleotide diversity, with ð values ranging from 0.17 to 0.94. All isolates showed significantly conserved regions (P>0.05). In conclusion, the observed variations in the sequences of the target bacterial strains can be attributed to resistance to antibiotics and gene transfer among bacterial strains in the hospital environment. Further sequence analyses of antibiotic resistance genes are warranted. Keywords: Multi-drug resistant bacteria, 16s rDNA sequencing, PCR, nucleotide diversity. A developed multidrug-resistant (MDR) hospital habitat microorganism for specific uses is part of understanding the mechanisms of the transfer of antibiotic resistance genes among different bacterial species. Isolation and identification of MDR microorganisms is challenging because these microorganisms exhibit highly specific characteristics that make them grow in their natural environments, such that they cannot be cultured using standard laboratory methods (Vijayan et al ., 2012). Competition among microbes for space and nutrients in the hospital environment represents a powerful selection pressure that can confer enhanced