A Simple Method for Direct Detection and Discrimination of &amp;lt;i&amp;gt;A. baumannii&amp;lt;/i&amp;gt; in Tracheal Aspirates without Culture Isolation

Cheguri H Swathi,Vemu Lakshmi,Sudhaharan Sukanya,Kamaraju Saipriya,Venkataraman Sritharan

doi:10.4236/aid.2020.102012

Abstract

Currently, available phenotyping and commercial methods report A. baumannii only as Acinetobacter calcoaceticus-baumannii complex (ACB) and do not identify individual members of the complex. This is a single blind study aimed to evaluate certain commonly used species-specific genetic markers namely, Intergenic Transcribed Spacer region in 16S rRNA of A. baumannii (Ab-ITS) and gyrB, for identification of ACB members. These molecular targets were first validated on clinical isolates (n = 200) and subsequently on uncultured tracheal aspirates (n = 172). Among the clinical isolates, 183/200 (91.5%) were positive for Ab-ITS. The clinical isolates 17 (17/200) which are failed to amplify in Ab-ITS PCR were subsequently diagnosed by gyrB PCR as A. calcoaceticus (n = 2), A. pitti (n = 6) and A. nosocomialis (n = 9) but not A. baumannii. Among the tracheal aspirates, 62 samples were reported as sterile in Advanced Expert System of VITEK-2, among the remaining 110 samples, 68.1% (75/110) samples contained Ab-ITS target. Twenty-five of the sterile samples (25/62) were found to contain Ab-ITS target sequence. Since, our sample processing method enabled identification of all the species of ACB complex by PCR even in uncultured tracheal aspirates, adaptation of our protocol would enable same day (6 - 8 h) reporting and help the clinician make evidence based therapeutic decision quickly.

Highlights

Acinetobacter baumannii causes a vast array of infections including ventilator associated pneumonia (VAP), secondary meningitis, urinary tract infections, septicemia and other severe infectious complications in the critical care (CCU) patients [1] [2]
The clinical isolates 17 (17/200) which are failed to amplify in Ab-intergenic spacer (ITS) polymerase chain reaction (PCR) were subsequently diagnosed by gyrB PCR as A. calcoaceticus (n = 2), A. pitti (n = 6) and A. nosocomialis (n = 9) but not A. baumannii
We present in this article our experience of using gyrB based target sequences for differentiation of individual members of Acinetobacter calcoaceticus-baumannii complex (ACB) complex

Summary

Introduction

Acinetobacter baumannii causes a vast array of infections including ventilator associated pneumonia (VAP), secondary meningitis, urinary tract infections, septicemia and other severe infectious complications in the critical care (CCU) patients [1] [2]. Clinical microbiology laboratories usually report A. baumannii as “ACB complex”. This includes six individual species namely A. baumannii, A. calcoaceticus, A. pitti, and A. nosocomialis, A. seifertii, A. lactucae [3]. These members have epidemiological and clinically relevant differences. Members of ACB complex pose limitations in phenotyping and conventional microbiological identification [5] [6] [7] [8]. It would be desirable to apply molecular methods to identify the individual species of the ACB complex [7] [9]. It would be ideal to be able to detect and report the species directly rapidly from the clinical sample without waiting for culture isolation and characterization

Methods

Results

Discussion

Conclusion