A Comparative study of the Phenotypic and Molecular Techniques for Identification of Pathogenic Gram-Negative Bacteria in Pharmaceutical Water

Abstract
Rapid and accurate identification of pathogenic bacteria is a fundamental goal in microbiological monitoring of pharmaceutical water quality, but one that is very difficult for many slow-growing and fastidious microorganisms. Purified water is widely used in pharmaceutical manufacturing in washing of equipment, preparation of finished product, and it is also used for rinsing equipment or for the preparation of disinfectants and detergents. A total of 150 purified water samples were collected from all the plant’s departments over a period of 3 months from December 2020 to February 2021. Each sample was filtered on 0.45µ membrane filter then cultured on R2A agar plates. Bacterial isolates from water samples were morphologically categorized based on shape, size, colour, and its characteristic growth on selective media and biochemical testing. Colonies with different colors and morphologies were subjected to amplification and sequencing of a 1000–1500 nt portion of the 16S rRNA gene. A total of 235 colonies were isolated. These were categorized into 29 isolates as colonies showing identical morphology were grouped together. 16S rRNA Sanger sequencing categorized these 29 isolates into 18 genera and 22 species. Sixty-six colonies (categorized into 7 isolates) were presumed to the genus Pseudomonas by conventional method. However, 16S rRNA Sanger sequencing indicated 21 out of these 66 colonies (categorized into 2 isolates) belonged to another genus. For the purified water system, Pseudomonas was the most common gram-negative genus isolated, followed by Enterobacter then Klebsiella and Citrobacter.