Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment

Shannon, Kelly; Lee, D.-Y.; Trevors, J. T.; Beaudette, Lee A.

doi:10.1016/j.scitotenv.2007.02.039

Cited by 219 publications

(148 citation statements)

References 18 publications

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“…Previous reports on the use of quantitative PCR for these pathogens include Studer et al (2008;S. aureus), Li and Drake (2001), Shannon et al (2007;E. coli) and Shannon et al (2007;K.…”

Section: Discussionmentioning

confidence: 99%

Effects of an immunomodulatory feed additive on the development of mastitis in a mouse infection model using four bovine-origin isolates

Rowson¹,

Wang²,

Aalseth

et al. 2011

Animal

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The goal of this study was to examine the ability of a commercially available feed additive (OmniGen-AF) to reduce mammary infections caused by a single strain of mastitic pathogens (Streptococcus uberis, Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae) and to examine the effects of the additive on markers of mammary immunity. Four experiments were completed using a murine model of bovine mastitis. Infection progression was examined using Sybr-green-and TaqMan-based quantitative PCR assays of 16S ribosomal DNA. Infection of the mammary gland with all pathogens caused rapid (24 to 48 h) appearance of pathogen DNA in mammary tissue. Provision of the feed additive for 2 weeks before infection significantly (P , 0.05) reduced the extent of pathogen DNA accumulation in models of S. uberis, E. coli and S. aureus infection. The additive was ineffective in reducing mammary infections caused by K. pneumoniae. We examined mechanisms of action of the additive through assessment of mammary concentrations of mammary myeloperoxidase (MPO), major histocompatibility complex 2 class II (MHC) and macrophage inflammatory protein-1a (MIP) messenger RNA (mRNA) concentrations and by examining serum complement C3 concentration. Infection of the mammary gland increased concentrations of MPO and MHC mRNAs (P , 0.05). Ability of the pathogen to elicit changes in mammary MPO and MHC gene expression was enhanced by the provision of the additive for 2 weeks before infection. These data imply that the additive increased the mammary inflammatory response and increased antigen presentation during a mammary infection. Value of the additive in preventing mastitis in cattle awaits additional studies using a bovine model and further evaluation of additional strains of the pathogens used in this study.

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“…Previous reports on the use of quantitative PCR for these pathogens include Studer et al (2008;S. aureus), Li and Drake (2001), Shannon et al (2007;E. coli) and Shannon et al (2007;K.…”

Section: Discussionmentioning

confidence: 99%

Effects of an immunomodulatory feed additive on the development of mastitis in a mouse infection model using four bovine-origin isolates

Rowson¹,

Wang²,

Aalseth

et al. 2011

Animal

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“…The potential of S. aureus to transfer genes encoding antibiotic resistance in wastewater has been shown (Ohlsen et al, 2003). The occurrence and survival of S. aureus in wastewater environments have not been well studied, but the indication from both cultivation and PCR is that S. aureus occurs at low levels in municipal wastewaters and surface waters ( [Schwartz et al, 2003], [Savichtcheva et al, 2007] and [Shannon et al, 2007]). In the present study S. aureus was detected to almost the same extent in OUT as well as IN (Table 2), and other groups have been able to isolate S. aureus from microbial aerosols in WWTPs (Fracchia et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies have shown that S. aureus has a low prevalence in hospital and municipal wastewaters ( [Schwartz et al, 2003] and [Shannon et al, 2007]). The mecA gene has been detected both in municipal and in hospital wastewaters (Börjesson et al, in press;[Schwartz et al, 2003] and [Volkmann et al, 2004]).…”

Section: Introductionmentioning

confidence: 99%

A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant

et al. 2009

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The spread of methicillin-resistant Staphylococcus aureus (MRSA), in which the mecA gene mediates resistance, threatens the treatment of staphylococcal diseases. The aims were to determine the effect of wastewater treatment processes on mecA gene concentrations, and the prevalence of S. aureus and MRSA over time. To achieve this a municipal wastewater treatment plant was investigated for the mecA gene, S. aureus and MRSA, using real-time PCR assays. Water samples were collected monthly for one year, at eight sites in the plant, reflecting different aspects of the treatment process. The mecA gene and S. aureus could be detected throughout the year at all sampling sites. MRSA could also be detected, but mainly in the early treatment steps. The presence of MRSA was verified through cultivation from inlet water. The concentration of the mecA gene varied between months and sampling sites, but no obvious seasonal variation could be determined. The wastewater treatment process reduced the mecA gene concentration in most months. Taken together our results show that the mecA gene, S. aureus and MRSA occur over the year at all sites investigated.

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“…Total bacterial density was determined using a Taqman assay, in which a 466-bp fragment of the 16S ribosomal RNA gene was amplified, as described previously (Nadkarni et al, 2002). P. aeruginosa density was determined using a Taqman assay, which amplified a 65-bp fragment of the regA gene, as described previously (Shannon et al, 2007;Rogers et al, 2010). Details of the relevant primers and probes used are shown in Supplementary Table S1.…”

Section: Dna Extractionmentioning

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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Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment

Cited by 219 publications

References 18 publications

Effects of an immunomodulatory feed additive on the development of mastitis in a mouse infection model using four bovine-origin isolates

Effects of an immunomodulatory feed additive on the development of mastitis in a mouse infection model using four bovine-origin isolates

A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant

Reducing bias in bacterial community analysis of lower respiratory infections

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