Optimization of PMA-qPCR forStaphylococcus aureusand determination of viable bacteria in indoor air

Abstract

Staphylococcus aureus may cause infections in humans from mild skin disorders to lethal pneumonia. Rapid and accurate monitoring of viable S. aureus is essential to characterize human exposure. This study evaluated quantitative PCR (qPCR) with propidium monoazide (PMA) to quantify S. aureus. The results showed comparable S. aureus counts between exclusively live cells and mixtures of live/dead cells by qPCR with 1.5 or 2.3 μg/mL PMA (P>.05), illustrating the ability of PMA-qPCR to detect DNA exclusively from viable cells. Moreover, qPCR with 1.5 or 2.3 μg/mL PMA performed optimally with linearity over 10 -10 CFU/mL (R ≥0.9), whereas qPCR with 10, 23 or 46 μg/mL PMA significantly underestimated viable counts. Staphylococcus aureus and total viable bacteria were further determined with PMA-qPCR (1.5 μg/mL) from 48 samples from a public library and two university dormitories and four from outside. Viable bacteria averaged 1.9×10 cells/m , and S. aureus were detected in 22 (42%) samples with a mean of 4.4×10 cells/m . The number of S. aureus and viable bacteria were positively correlated (r=.61, P<.005), and percentages of S. aureus relative to viable bacteria averaged 12-44%. The results of field samples suggest that PMA-qPCR can be used to quantify viable S. aureus cells.

Detection and Quantification of Viable but Non-culturable Campylobacter jejuni

“…Thus, VBNC cell count can be estimated by subtracting the number of culturable cells from the total viable cell count determined using PMA-qPCR. This technique has been used for the detection of different VBNC bacterial cells, such as Escherichia coli and Vibrio parahaemolyticus (Afari and Hung, 2018;Chang and Lin, 2018;Zhong and Zhao, 2018;Telli and Dogruer, 2019).…”

Airborne Staphylococcus aureus in different environments—a review

“…Although the public health problem related to pathogenic bacteria of Staphylococcus aureus species is not new (Duckworth and Jordens 1990; Lowy 1998), yet in the last decade, year after year, more and more scientific alarms and reports about the increasing epidemiological risk have been coming from all continents (Agostino et al 2017; Chang and Lin 2018; Denis 2017), especially in the context of acquiring by this species the genes of antibiotics resistance (Arias and Murray 2015; Epstein et al 2016; Nadimpalli et al 2018; Spellberg et al 2008; Tacconelli et al 2018). It has been estimated that complications in treatment refer, depending on the source, to 11–53% of S. aure us bacteremia (Keynan and Rubinstein 2013).…”

“…Therefore, the bacteria are particularly significant in the case of the people exposed environmentally, including hospitals and in such environments where there is a high probability of the occurrence of pathogenic and multiantibiotic-resistant strains, including the municipal wastewater treatment plants (WWTP) (Boopathy 2017; Gordon and Lowy 2008) or animal farms (Friese et al 2013). The airborne transmission is as significant as the direct transmission of bacteria by the hands or mouth, although it may be difficult to indicate which of those routes is the most important for health care personnel (Bos et al 2016; Chang and Lin 2018). S. aureus bacteria, similarly to other species from Staphylococci genus, are characterized by a high survival rate in dry environments, e.g.…”

Simultaneous quantitative detection of viable Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. using sodium deoxycholate-propidium monoazide with multiplex real-time PCR

“…However, mRT-PCR alone cannot distinguish between dead and viable bacteria, which can lead to false-positive results. To eliminate the interference of DNA from dead cells, a strategy that relies on sodium deoxycholate (SD) combined with propidium monoazide (PMA) pretreatment had been reported (Chang and Lin, 2018;Huang et al, 2018). Sodium deoxycholate is a bile salt that promotes membrane rupture of dead cells; PMA is a nucleic acid cross-linking agent that can selectively penetrate bacteria with injured cell membranes to sequester the DNA, preventing its amplification during PCR (Liu and Mustapha, 2014;Erkus et al, 2016).…”