Rapid ultrasensitive diagnosis of pneumonia caused by Acinetobacter baumannii using a combination of enrichment and phage-based qPCR assay

Abstract

Background Accurate and rapid identification of ventilator associated or nosocomial pneumonia caused by Acinetobacter baumannii ( A. baumannii ) could improve the treatment.Methods In current study, we developed a phage-based real-time quantitative PCR (qPCR) combined with enrichment culture for rapid and specific detection of viable A. baumannii in sputum from lung infections. Through short-term plate incubation, bacteria can be enriched and the DNA polymerase reaction disturbance of sputum can be decreased greatly. This approach is based on detecting phage replication in live A. baumannii cells through Taqman qPCR.Results Through the built detection system, down to 1 CFU of A. baumannii can be detected within 6 h in spiked sputum samples without any steps of bacteria isolation and DNA extraction. The established method was then applied to detecting both A. baumannii in simulated sputum with 100% agreement with the spiked amount of the bacteria and one clinical sputum sample from an 80-year-old male lung infection patient caused by A. baumannii with perfect accuracy, demonstrating that the assay developed in this study has the merits of high rapidity, high sensitivity, good specificity and being able to detect live bacteria not dead bacteria.Conclusions The assay is a potentially clinical method for diagnosis of bacterial pneumonia infection caused by A. baumannii or other bacterial infection in sputum or complicated samples through switching to other types of phages.

The Use of Bacteriophages in Biotechnology and Recent Insights into Proteomics

“…Similar methods have demonstrated a change in redox reactions in Salmonella enterica, S. typhi and S. paratyphi after phage infection [92]. Luo and colleagues successfully used a combination of enrichment and phage-based qPCR assays to rapidly (in 6 h) detect Acinetobacter baumannii in sputum samples from patients with lung infections, using p53 phages [93]; similarly, it only took 10 h for Garrido-Maestu and coworkers to detect Salmonella enteritidis in chicken flesh [94,95]. Phage amplification procedures described in the literature include: (i) Detection of E. coli by a technique using antibody-conjugated beads to isolate amplified MS2 phages [96], and (ii) pathogenic E. coli and Salmonella Newport identification in food samples by using a procedure involving phage coated paper dipsticks and qPCR-mediated detection of viral progeny [97].…”

Bacteriophage-based bioassays: an expected paradigm shift in microbial diagnostics

“…This approach offers a promising avenue for the timely and accurate diagnosis of A. baumannii infections, particularly in the context of complex clinical samples such as sputum. The combination of qPCR and enrichment culture addresses both sensitivity and specificity concerns, providing a robust and efficient means of detecting this clinically significant pathogen [ 77 ].…”

Advances in the Development of Phage-Based Probes for Detection of Bio-Species

“…For example, a novel method PAA-qPCR (phage amplification combines with qPCR) was reported to detect Salmonella enteritidis in chicken meat samples [ 70 ]. Similarly, Acinetobacter baumannii has recently been detected using phage-based qPCR method [ 71 ].…”