Use of Sanger Sequencing in Etiological Diagnostics of Bacterial Complications in Hospital Environment

Abstract

Background. The precise identification of microorganism species that cause infectious complications in hospitalized patients is beyond doubt relevant in modern healthcare. The aim of this study was to determine the possibility of using Sanger sequencing in routine microbiological examination of patients in an Internal Medicine Clinic to improve the quality of etiological diagnosis of bacterial complications. Material and methods. Clinical isolates of microorganisms isolated from patients of a multidisciplinary medical center were studied. The study used classical microbiological methods of seeding and identification of cultures, as well as Sanger sequencing. Results. Sanger identification using the MicroSeq system (Applied Biosystems, USA) ensured identification of all 231 studied bacterial isolates – causative agents of nosocomial infections. For differential diagnostics of streptococci and coagulase-negative staphylococci, in some cases, when the known sequences of the first 500 nucleotides of the 16S rRNA gene of two species differed by 1–2 nucleotides, increasing the discrimination level of species identification to 100% allowed valid determination of the species affiliation of the studied microorganism. Phenotypic methods failed to identify a significant proportion of species (25.9%) of nosocomial infection pathogens, and only 8 (13.8%) of them were reliably identified in all cases. The use of Sanger sequencing to identify bacteria led to a long-term effect associated with improved qualifications of laboratory doctors, and enhanced discriminatory capabilities of visual assessment of the macromorphology of bacterial cultures, which is important for identifying all types of microorganisms present in biosubstrates. Conclusion. The Sanger sequencing method is highly efficient and quite cost effective, compared to the biochemical test panels widely used in clinical practice — the «gold standard» method in the etiological diagnosis of bacterial complications in the clinic of internal diseases.

Keywords

• species identification
• 16s rrna gene
• hypervariable regions
• microseq
• phenotypic methods
• molecular methods
• maldi-tof mass spectrometry
• nosocomial
• hospital-acquired
• healthcare-associated infections
• postgraduate education