Molecular Viability Testing of Bacterial Pathogens from a Complex Human Sample Matrix

Kris M Weigel,Jae-Hyun Chung,Kelly L Jones,Gerard A Cangelosi,Christian Valcke,Julie S Do,Jody Melton Witt,Riccardo Manganelli

doi:10.1371/journal.pone.0054886

Kris M Weigel, Jae-Hyun Chung + Show 6 more

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https://doi.org/10.1371/journal.pone.0054886

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Journal: PLoS ONE	Publication Date: Jan 24, 2013
Citations: 29	License type: CC BY 4.0

Affiliation: University of Washington, Novartis (United States)

Abstract

Assays for bacterial ribosomal RNA precursors (pre-rRNA) have been shown to distinguish viable from inactivated bacterial cells in drinking water samples. Because the synthesis of pre-rRNA is rapidly induced by nutritional stimulation, viable bacteria can be distinguished from inactivated cells and free nucleic acids by measuring the production of species-specific pre-rRNA in samples that have been briefly stimulated with nutrients. Here, pre-rRNA analysis was applied to bacteria from serum, a human sample matrix. In contrast to drinking water, serum is rich in nutrients that might be expected to mask the effects of nutritional stimulation. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were used to detect pre-rRNA of four bacterial species: Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and the Mycobacterium tuberculosis complex. These species were chosen for their clinical significance and phylogenetic diversity (Proteobacteria, Firmicutes, and Actinobacteria). To maximize resolving power, pre-rRNA was normalized to genomic DNA of each pathogen. When viable cells were shifted from serum to bacteriological culture medium, rapid replenishment of pre-rRNA was always observed. Cells of P. aeruginosa that were inactivated in the presence of serum exhibited no pre-rRNA response to nutritional stimulation, despite strong genomic DNA signals in these samples. When semi-automated methods were used, pre-rRNA analysis detected viable A. baumannii cells in serum at densities of ≤100 CFU/mL in <5.5 hours. Originally developed for rapid microbiological analysis of drinking water, ratiometric pre-rRNA analysis can also assess the viability of bacterial cells derived from human specimens, without requiring bacteriological culture.

Highlights

Bacterial pathogens such as Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium species, are common causes of healthcare-associated infections
This study asked whether ratiometric pre-ribosomal RNA (rRNA) analysis, which was originally developed for analysis of drinking water, could assess the viability of bacterial cells in human specimens
Human fluids such as serum are rich in certain nutrients, they are usually limited in some required nutrients, and bacterial growth rates are sub-maximal in such environments

Summary

Introduction

Bacterial pathogens such as Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium species, are common causes of healthcare-associated infections. Laboratory diagnosis of such infections must balance the needs of sensitivity, speed, and clinical relevance. Bacterial mRNA is unstable and difficult to detect [11], while rRNA can persist in dead bacterial cells [1] Another approach involves treating bacteria with DNA intercalators that penetrate inactivated cells and inhibit PCR amplification, but are excluded from viable cells [12,13]. These methods are effective but performance varies with sample conditions [14,15,16,17]

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