Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

Taşkın, Bilgin; Gözen, Ayşe Gül; Duran, Metin

doi:10.1128/aem.02895-10

Cited by 97 publications

(74 citation statements)

References 33 publications

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“…Enterobacteriaceae bacteria also serve as indicators of environmental pollution and can induce bacteremia (20). Thus, Enterobacteriaceae bacteria in milk have been targeted as a topic of broad concern.…”

Section: Discussionmentioning

confidence: 99%

“…As a control compound for LD discrimination, 20 mM PMA (Biotium Inc., Hayward, CA, USA) was diluted in SW in the dark to concentrations of 100, 1,000, 2,500, 5,000, and 10,000 M. The diluted PMA solutions (10 l) were added to E. coli or C. sakazakii suspensions (generally, 90 l was used, with the exception that a 970-l suspension was used for milk samples) under a safelight. The bacterial suspensions were uniformly mixed, and the tubes were kept at 5°C for 10 min (typical treatment conditions) under a safelight, followed by visible-light irradiation (5 min) (19,20).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Innovative Use of Palladium Compounds To Selectively Detect Live Enterobacteriaceae in Milk by PCR

Soejima

Iwatsuki

2016

Appl Environ Microbiol

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Ethidium monoazide and propidium monoazide (EMA and PMA) have been used in combination with PCR for more than a decade to facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving lower limits of detection and quantification of targeted live cells, fewer laborious procedures, lower costs, and potentially higherthroughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry costs that are 3 times higher because of the requirement for much larger amounts for LD discrimination than palladium compounds. Palladium compounds can penetrate dead (compromised) but not live bacteria and can be chelated primarily by chromosomal DNA and cell wall transmembrane proteins, with small amounts of DNA-binding proteins in vivo. The new mechanism for palladium compounds is obviously different from that of platinum compounds, which primarily target DNA. Combining palladium compounds with PCR (Pd-PCR) in water resulted in discrimination between live and dead Enterobacteriaceae bacteria that was much clearer than that seen with the PMA method. Pd-PCR correlated with reference plating or with the currently used PMA-PCR method for pasteurized milk, based on EN ISO 16140:2003 validation. Pd-PCR enabled us to specifically detect and assay viable Enterobacteriaceae cells at concentrations of 5 to 10 CFU/ml in milk while following U.S./EU regulations after a 4.5-h process in a typical laboratory exposed to natural or electric light, as specified by U.S./EU regulations. IMPORTANCEEthidium monoazide and propidium monoazide (EMA and PMA) facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving fewer laborious procedures, lower costs, and potentially higherthroughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry costs that are 3 times higher because of the requirement for much larger amounts for LD discrimination than palladium compounds, which have also a novel reaction mechanism different from that of platinum compounds. In view of testing cost, palladium compounds are also very useful here compared with platinum compounds. Ultimately, the innovative Pd-PCR method may be also substituted for the currently used reference plating methods. The PCR is a widely available tool that is used to detect bacteria and viruses in food and in environmental and clinical samples. However, PCR cannot distinguish live from dead bacteria. During reverse transcription-PCR targeting mRNA, a high concentration of contaminating dead bacteria (4 log 10 to 7 log 10 cells/ml) will trigger a false-positive result owing to t...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Innovative Use of Palladium Compounds To Selectively Detect Live Enterobacteriaceae in Milk by PCR

Soejima

Iwatsuki

2016

Appl Environ Microbiol

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“…PMA treatment for this purpose has been shown to be effective in a range of contexts (Nocker et al, 2007a;Bae and Wuertz, 2009;Kralik et al, 2010;Nam et al, 2011;Taskin et al, 2011), including the assessment of microbiota present in CF airway samples (Rogers et al, 2008(Rogers et al, , 2010. Further, this strategy has been used previously in conjunction with bacterial pyrosequencing in waste-water treatment systems .…”

Section: Introductionmentioning

confidence: 99%

Reducing bias in bacterial community analysis of lower respiratory infections

et al. 2012

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High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved accuracy and depth of characterisation of lower respiratory infections. However, such approaches suffer from an inability to distinguish between DNA derived from viable and non-viable bacteria. This discrimination represents an important step in characterising microbial communities, particularly in contexts with poor clearance of material or high antimicrobial stress, as non-viable bacteria and extracellular DNA can contribute significantly to analyses. Pre-treatment of samples with propidium monoazide (PMA) is an effective approach to non-viable cell exclusion (NVCE). However, the impact of NVCE on microbial community characteristics (abundance, diversity, composition and structure) is not known. Here, adult cystic fibrosis (CF) sputum samples were used as a paradigm. The effects of PMA treatment on CF sputum bacterial community characteristics, as analysed by pyrosequencing and enumeration by species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR, were assessed. At the local community level, abundances of both total bacteria and of P. aeruginosa were significantly lower in PMA-treated sample portions. Meta-analysis indicated no overall significant differences in diversity; however, PMA treatment resulted in a significant alteration in local community membership in all cases. In contrast, at the metacommunity level, PMA treatment resulted in an increase in community evenness, driven by an increase in diversity, predominately representing rare community members. Importantly, PMA treatment facilitated the detection of both recognised and emerging CF pathogens, significantly influencing ‘core’ and ‘satellite’ taxa group membership. Our findings suggest failure to implement NVCE may result in skewed bacterial community analyses.

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“…Morphologically damaged oocysts and cells have unprotected nucleic acid. Therefore, their amplification is restricted after EMA and PMA photo-activation (Nocker et al, 2006;Hein et al, 2007;Brescia et al, 2009;Chen and Chang, 2010;Byappanahalli et al, 2010;Taskin et al, 2011).…”

Section: Dye Based Viability Pcrmentioning

confidence: 99%

“…It has been demonstrated that Propidium Monoazide (PMA) (Biotium, Hayward, CA), a DNA intercalating dye, combined with PCR/qPCR methods can be used for selective detection/quantification of viable bacteria, protozoa and viruses from wastewater, sludge and environmental samples (Fittipaldi et al, 2011;Taskin et al, 2011;Nkuipou-Kenfack et al, 2013;Alonso et al, 2014;Li et al, 2014;Gensberger et al, 2014;Santiago et al, 2015). The working mechanism of PMA is that it penetrates non-viable cells or oo(cysts), and makes a covalent bond with DNA upon exposure to light preventing PCR amplification (Nocker et al, 2006;Nocker et al, 2007).…”

Section: General Introductionmentioning

confidence: 99%

Detection of viable hookworm ova from wastewater and sludge

Gyawali¹

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Infectious helminths are a worldwide major public health problem. In terms of morbidity, approximately 3 × 10 9 people are infected by soil-transmitted helminths (STHs) influencing rates of malnutrition and failure to thrive. All of this increases the global disease burden (GDB) by up to 5.9 × 10 6 Daily Adjusted Life Years (DALYs). Helminth infections are more often seen as a major public health issue for developing countries however, concerns have been expressed in many of the developed nations because of the increased use of treated wastewater and sludge with no clear way of knowing the infection potential that can be attributed from such water.Guidelines have been established to minimise the potential public health risk associated with wastewater and sludge reuse. For example, the WHO guideline specified ≤ 1 ova (Ascaris lumbricoides) per L liquid (wastewater) or 4 g dry solid (sludge) for unrestricted use. Various wastewater treatment processes have been recommended to remove the helminths ova from wastewater depending upon its reuse. However, detection methods used to quantify viable helminths ova from wastewater has limitations. Therefore there is always a potential public health risk associated with reuse of wastewater and sludge.In this research, a real-time PCR method was developed. The new PCR method is rapid and specific. The method was found to be able to detect less than one ovum in one litre treated wastewater and approximately four ova in one litre raw wastewater and ~ 4 g sludge.The real-time PCR method was modified to a quantitative PCR (qPCR) method and further used to quantify hookworm ova from wastewater and sludge. The qPCR had estimated an average of 1.1, 8.6 and 67.3 ova for treated wastewater that was seeded with 1, 10 and 100 ova, respectively. The gene copy numbers obtained for 1, 10 and 100 ova by qPCR varied significantly (P < 0.05) within the tested samples indicating that absolute quantification of ova may not be accurate. Despite the difficulty quantifying accurate numbers of hookworm ova, the lower limit of quantification (LLOQ) of the qPCR method was 30 gene copies. This was a lot less than the gene copies produced by one ovum. Therefore, the qPCR method has potential to use for complying with wastewater guidelines.. iiAlthough the overall aim of this research was to develop a sensitive and specific method for quantitative detection of viable hookworm ova from wastewater, the importance of recovering the ova from wastewater and sludge samples for accurate detection was identified. While determining appropriate recovery rates was not an aim of this research, a suitable method that could be used to standardise research outcomes for further study was established. Therefore, ova recovery rate by different rapid methods was evaluated for further experiments. The result indicated that the ova recovery rate was higher for the treated wastewater (0.2 -50%) than the raw wastewater (0.3 -35%) and sludge (0.02 -4.7%) samples. A significant difference (P < 0.05) was observed between...

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Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

Cited by 97 publications

References 33 publications

Innovative Use of Palladium Compounds To Selectively Detect Live Enterobacteriaceae in Milk by PCR

Innovative Use of Palladium Compounds To Selectively Detect Live Enterobacteriaceae in Milk by PCR

Reducing bias in bacterial community analysis of lower respiratory infections

Detection of viable hookworm ova from wastewater and sludge

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