Use of a Bacteroides thetaiotaomicron-specific α-1-6, mannanase quantitative PCR to detect human faecal pollution in water

Aims: The aims of this work were to develop a quantitative test, based on Bacteroides thetaiotaomicron, for human faecal pollution in water and to evaluate test performance. Methods and Results: qPCR primers, based on the complete genomic sequence of B. thetaiotaomicron VPI 5482, were designed and tested. The single-copy putative mannanase homologue, α-1-6 mannanase, was selected as the particular target and sequences within this gene chosen as the qPCR primers by Blast search for specificity to B. thetaiotaomicron. The average concentration of B. thetaiotaomicron in human faeces was 1·39 × 10⁸ cells per gram faeces and the detection limit was 9·3 B. thetaiotaomicron copies per qPCR procedure. Comparison of B. thetaiotaomicron content in sewage vs pooled nonhuman faecal samples indicated that the current assay is specific for sewage. Conclusion: The subject assay is potentially useful for quantification of sewage pollution in water. Significance and Impact of the Study: Bacteroides-associated markers, proposed for faecal source tracking, have exclusively been based on gene sequences related to generally classified and uncultured bacteria. However, genes associated with host-microbe interaction have been suggested as more specific markers. The present assay targets such a gene of B. thetaiotaomicron which is considered to be a symbiont in the human gut.     

Abundance and Diversity of Sulfur-Oxidizing Bacteria along a Salinity Gradient in Four Qinghai-Tibetan Lakes,China

Sulfur-oxidizing bacteria (SOB) play important roles in the sulfur cycle and are widespread in a number of environments, but their occurrence and relationship to geochemical conditions in (hyper)saline lakes are still poorly understood. In this study, the abundance and diversity of SOB populations were investigated in four Qinghai-Tibetan lakes (Erhai Lake, Gahai Lake 1, Gahai Lake 2 and Xiaochaidan Lake) by using quantitative polymerase chain reaction (qPCR) and soxB gene- (encoding sulfate thiohydrolase) based phylogenectic analyses. qPCR analyses showed that in the studied lakes, the total bacterial 16S rRNA and soxB gene abundances in the sediments were distinctly higher than in the overlying waters. The 16S rRNA gene abundance in the waters ranged 5.27 × 10⁶–6.09 × 10⁸ copies per mL and 7.39 × 10¹⁰–2.9 × 10¹¹ copies per gram sediment. The soxB gene abundance in the waters ranged from 1.88 × 10⁴ to 5.21 × 10⁵ per mL and 4.73 × 10⁶–2.65 × 10⁷ copies per gram sediment. The soxB gene in the waters of the two hypersaline lakes (Gahai Lake 2 and Xiaochaidan Lake) was more abundant (2.97 × 10⁵ and 5.21 × 10⁵ copies per mL) than that in the two low-salinity lakes (1.88 × 10⁴ and 3.36 × 10⁴ copies per mL). Phylogenetic analysis showed that Alpha- and Betaproteobacteria were dominant SOB in the investigated lakes, and the composition of proteobacterial subgroups varied with salinity: in freshwater Erhai Lake and low-salinity Gahai Lake 1, the SOB populations were dominated by the Betaproteobacteria, whereas in hypersaline Lake Gahai 2 and Xiaochaidan Lake, the SOB populations were dominated by Alphaproteobacteria. Overall, salinity played a key role in controlling the diversity and distribution of SOB populations in the investigated Qinghai-Tibetan lakes.     

Metalworking fluids biodiversity characterization

Aims: Hypersensitivity pneumonitis of machinists associated with metalworking fluids (MWF) was recently linked to Mycobacterium immunogenum. In addition to Mycobacterium, impacts of continuous and massive contact to other micro‐organisms, such as Pseudomonas, were little studied. This report intended to quantify and characterize the microbial load of 44 in‐use MWF. Methods and Results: The main biodiversity of MWF was assessed using cultural methods, quantitative PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE). Total bacteria concentrations ranged from undetectable to 10⁹ 16S rRNA gene copies per millilitre. Concentrations obtained by qPCR were up to five orders of magnitude higher than by culture, suggesting that MWF contamination is generally underestimated. Two samples showed high concentrations of Myco. immunogenum (1·55 × 10⁷ and 3·49 × 10⁵ 16S rRNA gene copies per millilitre). The overall biodiversity was low, as observed by culture and DGGE, and was comparable to data found in the literature. Pseudomonas pseudoalcaligenes was by far the main bacteria found in MWF samples (33 out of 44), followed by Ochrobactrum anthropi (32 out of 44). There was no significant relationship between the biodiversity profiles and the kind of MWF or equipment used, making it difficult to predict which micro‐organisms will colonize each particular MWF. Conclusions: Very high concentrations of bacteria were found in most MWF studied and limited biodiversities were observed. Many species of micro‐organisms were retrieved from MWF samples, but they were mostly colonized by Pseudomonas pseudoalcaligenes and Ochrobactrum anthropi. Significance and Impact of the Study: The major micro‐organisms observed or recovered in this study from in‐use MWF were present in very high concentrations, and thus further studies are needed to confirm their role in workers’ respiratory disorders or health‐related problems.     

Diversity and quantity of ammonia-oxidizing Archaea and Bacteria in sediment of the Pearl River Estuary, China

The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the sediment of the Pearl River Estuary were investigated by cloning and quantitative real-time polymerase chain reaction (qPCR). From one sediment sample S16, 36 AOA OTUs (3% cutoff) were obtained from three clone libraries constructed using three primer sets for amoA gene. Among the 36 OTUs, six were shared by all three clone libraries, two appeared in two clone libraries, and the other 28 were only recovered in one of the libraries. For AOB, only seven OTUs (based on 16S rRNA gene) and eight OTUs (based on amoA gene) were obtained, showing lower diversity than AOA. The qPCR results revealed that AOA amoA gene copy numbers ranged from 9.6 × 10⁶ to 5.1 × 10⁷ copies per gram of sediment and AOB amoA gene ranged from 9.5 × 10⁴ to 6.2 × 10⁵ copies per gram of sediment, indicating that the dominant ammonia-oxidizing microorganisms in the sediment of the Pearl River Estuary were AOA. The terminal restriction fragment length polymorphism results showed that the relative abundance of AOB species in the sediment samples of different salinity were significantly different, indicating that salinity might be a key factor shaping the AOB community composition.     

Phylogenetic analysis of Bacteroidales 16S rRNA gene sequences from human and animal effluents and assessment of ruminant faecal pollution by real‐time PCR

Aims: The aims of this study were to evaluate the host‐specific distribution of Bacteroidales 16S rRNA gene sequences from human‐ and animal‐related effluents and faeces, and to define a ruminant‐specific marker. Methods and Results: Bacteroidales 16S rRNA gene clone libraries were constructed from samples of effluent (sewage, bovine manure and pig slurry) and faeces (human, bovine, pig and wild bird), using PCR primers targeting order Bacteroidales. The phylogenetic analysis revealed six main distinct human‐, bovine‐, pig‐ and wild bird‐specific clusters. From the bovine‐specific cluster II, we designed a ruminant‐specific marker, Rum‐2‐Bac, and this showed 97% sensitivity (n = 30) and 100% specificity (n = 40) when tested by TaqMan^® real‐time PCR. Average concentrations of this marker in bovine and sheep faeces and in bovine manure were 8·2 ± 0·5, 8·4 ± 1·3 and 7 ± 0·5 log₁₀ copies per gram, respectively. It was also quantified in samples of runoff water impacted by bovine manure, with average concentrations of 5·1 ± 0·3 log₁₀ copies per millilitre water. Conclusions: Our results confirmed that some members of Bacteroidales isolated from effluents and faeces had host‐specific distributions. Identification of a bovine‐specific cluster made it possible to design a reliable ruminant‐specific marker. Significance and Impact of the Study: The host‐specific distribution of Bacteroidales sequences from effluents mirrored the host‐specific distribution of sequences observed in individual faeces. This efficient new ruminant‐specific Bacteroidales 16S rRNA marker represents a useful addition to the microbial source tracking toolbox.     

Quantification of host-specific <Emphasis Type="Italic">Bacteroides–Prevotella</Emphasis> 16S rRNA genetic markers for assessment of fecal pollution in freshwater

Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides–Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9–11 log₁₀ copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides–Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r ² = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50–800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments.     

The presence of atrazine and atrazine-degrading bacteria in the residential, cattle farming, forested and golf course regions of Lake Oconee

Aims: To assess the concentration of atrazine in Lake Oconee and develop a qPCR assay as a potential marker for the presence of atrazine‐degrading bacteria indicating atrazine contamination. Methods and Results: Water and sediment samples were collected from the Oconee Lake at four golf course sites, two residential sites, one cattle farming site and a forested site. Atrazine concentration at the study sites was determined using an ELISA kit and indicated the presence of atrazine from 0·72 ppb at the forested sites to 1·84 ppb at the golf course sites. QPCR results indicate the presence of atzA gene (atrazine chlorohydrolase) from 1·51 × 10² gene copies at the residential sites to 3·31 × 10⁵ gene copies per 100 ml of water at the golf course regions of the lake and correlated (r = 0·64) with atrazine concentration. Sediment samples had higher atzA gene copies compared with the water samples (P < 0·05). Conclusions: Atrazine concentration and the highest quantity of atzA gene were detected in the golf course regions of the lake. Overall, atrazine concentration monitored in Lake Oconee was below the Environment Protection Agency (EPA) regulatory standards. Significance and Impact of the Study: Quantitative PCR is an efficient technique for assessing the presence of atrazine catabolism gene as a functional marker for atrazine‐degrading bacteria and the presence of atrazine contamination.     

Quantitative real‐time PCR detection of a harmful unarmoured dinoflagellate,Karlodinium australe (Dinophyceae)

We investigated a harmful algal bloom (HAB) associated with the massive fish kills in Johor Strait, Malaysia, which recurred a year after the first incident in 2014. This incident has urged for the need to have a rapid and precise method in HAB monitoring. In this study, we develop a SYBR green‐based real‐time PCR (qPCR) to detect the culpable dinoflagellate species, Karlodinium australe. Species‐specific qPCR primers were designed in the gene region of the second internal transcribed spacer of the ribosomal RNA gene (rDNA). The species specificity of the primers designed was evaluated by screening on the non‐target species (Karlodinium veneficum, Takayama spp., and Karenia spp.) and no cross‐detection was observed. The extractable gene copies per cell of K. australe determined in this study were 19 998 ± 505 (P < 0.0001). Estimation of cell densities by qPCR in the experimental spiked samples showed high correlation with data determined microscopically (R² = 0.93). Using the qPCR assay developed in this study, we successfully detected the 2015 bloom species as K. australe. Single‐cell PCR and rDNA sequencing from the field samples further confirmed the finding. With the sensitivity as low as five cells, the qPCR assay developed in this study could effectively and rapidly detect cells of K. australe in the environmental samples for monitoring purpose.     

A high‐throughput and quantitative hierarchical oligonucleotide primer extension (HOPE)‐based approach to identify sources of faecal contamination in water bodies

As faecal contamination of recreational and drinking water impairs the water quality and threatens public health, water bodies are routinely monitored for faecal coliforms to detect contamination. However, faecal coliforms are facultative anaerobes that survive and reproduce in ambient waters, and their presence does not depict the origin of contamination. Therefore, the use of Bacteroides‐Prevotella 16S rRNA gene to perform faecal source tracking has been proposed and applied. Here, we demonstrate the use of a new molecular method termed hierarchical oligonucleotide primer extension (HOPE) to simultaneously detect human‐associated Bacteroides spp. and three clusters of cow‐, pig‐ and dog‐specific uncultivated Bacteroidales. The method correctly identifies the origin of faecal contamination when tested against human, cow, pig and dog faeces (n = 17, 17, 16 and 13 respectively), and in waters contaminated with faeces of known origins. Subsequent tests with a total of 21 blind samples show that HOPE is able to accurately indicate single or multiple sources of faecal contamination originating from pigs, cows and humans in 81% of the blind samples. HOPE can further correctly detect and identify faecal contamination in five sampling sites located along a canal in southern Taiwan, and the results are validated against conventional faecal coliform tests and quantitative PCR. Overall, this study demonstrates HOPE as a quantitative and high‐throughput method that can identify sources of faecal contamination.     

Characterization and validation of a chemiluminescent assay based on a 1,2-dioxetane for rapid detection of enterococci in contaminated water and comparison with standard methods and qPCR

Aims: To evaluate the potential for using a novel chemiluminescence‐based enzyme assay for rapid detection of enterococci in water contaminated with faecal waste. Methods and Results: The novel assay (EntLight) was based on the enzymatic hydrolysis of the chemiluminescent 1,2‐dioxetane [(4‐methoxy‐4(3‐β‐d ‐glucoside‐4‐chlorophenyl)]spiro[1,2‐dioxetane‐3‐1,3‐tricyclo[7·3·1·0^2,7]tridec‐2,7‐ene] specific for β‐d ‐glucosidase. The specificity of the proposed EntLight assay was characterized using 26 different Enterococcus strains and 10 bacterial genera other than Enterococcus. With an analysis time of ≤8 h, the assay was found to be sensitive and specific. Validation experiments were carried out using water samples contaminated with raw municipal wastewater in comparison with qPCR and ISO standard methods. EntLight was successfully applied to detect enterococci in contaminated water within ≤8 h, and the proposed assay correlated well with both qPCR and ISO standard methods (R² > 0·776). Conclusions: EntLight can be applied to rapid and simple detection of viable enterococci in water contaminated with faecal matter. Significance and Impact of the Study: The novel EntLight assay and qPCR have the potential to be used as methods for early warning (1–7 h) of faecal pollutions in different water types.     

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis

Aim: To develop a TaqMan probe‐based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay’s specificity, detection limit, intra‐ and inter‐assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100‐fold more sensitive than the cPCR. No cross‐reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10¹¹ and 2·75 × 10⁸ target copies ml^?1 of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.     

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR‐based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain‐specific quantitative real‐time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA‐intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10⁵–10⁹ cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g^?1 [mean (±SD)] of BbrY was detected in faeces by using strain‐specific transgalactosylated oligosaccharide–carbenicillin (T‐CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g. Conclusions: Strain‐specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain‐specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.     

Bacterial Community Structure in Two Permafrost Wetlands on the Tibetan Plateau and Sanjiang Plain,China

Permafrost wetlands are important methane emission sources and fragile ecosystems sensitive to climate change. Presently, there remains a lack of knowledge regarding bacterial communities, especially methanotrophs in vast areas of permafrost on the Tibetan Plateau in Northwest China and the Sanjiang Plain (SJ) in Northeast China. In this study, 16S rRNA-based quantitative PCR (qPCR) and 454 pyrosequencing were used to identify bacterial communities in soils sampled from a littoral wetland of Lake Namco on the Tibetan Plateau (NMC) and an alluvial wetland on the SJ. Additionally, methanotroph-specific primers targeting particulate methane monooxygenase subunit A gene (pmoA) were used for qPCR and pyrosequencing analysis of methanotrophic community structure in NMC soils. qPCR analysis revealed the presence of 10¹⁰ 16S rRNA gene copies per gram of wet soil in both wetlands, with 10⁸ pmoA copies per gram of wet soil in NMC. The two permafrost wetlands showed similar bacterial community compositions, which differed from those reported in other cold environments. Proteobacteria, Actinobacteria , and Chloroflexi were the most abundant phyla in both wetlands, whereas Acidobacteria was prevalent in the acidic wetland SJ only. These four phyla constituted more than 80 % of total bacterial community diversity in permafrost wetland soils, and Methylobacter of type I methanotrophs was overwhelmingly dominant in NMC soils. This study is the first major bacterial sequencing effort of permafrost in the NMC and SJ wetlands, which provides fundamental data for further studies of microbial function in extreme ecosystems under climate change scenarios.     

Correlation of quantitative PCR for a poultry-specific brevibacterium marker gene with bacterial and chemical indicators of water pollution in a watershed impacted by land application of poultry litter

The impact of fecal contamination from human and agricultural animal waste on water quality is a major public health concern. Identification of the dominant source(s) of fecal pollution in a watershed is necessary for assessing the safety of recreational water and protecting water resources. A field study was conducted using quantitative PCR (qPCR) for the 16S rRNA gene of Brevibacterium sp. LA35 to track feces-contaminated poultry litter in environmental samples. Based on sensitivity and specificity characteristics of the qPCR method, the Bayesian conditional probability that detection of the LA35 marker gene in a water sample represented a true-positive result was 93%. The marker's covariance with fecal indicator bacteria (FIB) and metals associated with poultry litter was also assessed in litter, runoff, surface water, and groundwater samples. LA35 was detected in water and soil samples collected throughout the watershed, and its concentration covaried with concentrations of Escherichia coli, enterococci, As, Cu, P, and Zn. Significantly greater concentrations of FIB, As, Cu, P, and Zn were observed in edge-of-field runoff samples in which LA35 was detected, compared to samples in which it was not detected. Furthermore, As, Cu, P, and Zn concentrations covaried in environmental samples in which LA35 was detected and typically did not in samples in which the marker gene was not detected. The covariance of the poultry-specific LA35 marker gene with these known contaminants from poultry feces provides further evidence that it is a useful tool for assessing the impact of poultry-derived fecal pollution in environmental waters.     

Methicillin-resistant Staphylococcus aureus ST398 contamination and transmission in pigs after a low dose inoculation

Aims: Methicillin‐resistant Staphylococcus aureus (MRSA) ST398 has recently been described as a zoonotic agent. Its transmission between animals seems to be a pivotal factor in its emergence and dissemination. This experimental trial was performed to describe MRSA ST398 contamination and transmission in pigs after a low dose inoculation. Methods and results: Twelve specific pathogen‐free (SPF) pigs were randomly divided between two separate pens. Three pigs in each pen received a nasal inoculation of 2 × 10⁴ colony‐forming units per animal, and three naïve pigs were left in contact with them. Every 2 days and at necropsy, different samples were screened for MRSA. It was detected in nasal swabs from five inoculated and three naïve contact pigs, as early as 1 day after inoculation. MRSA was also found in environmental wipes but never in faecal samples. At necropsy, MRSA was detected in the lymph nodes of two contact pigs and in the tonsils and lymph nodes of three inoculated pigs. Twelve other SPF pigs were included as negative control in a separate room. Conclusion: This experiment showed that inoculation of a low dose of MRSA ST398 could lead to the horizontal transmission of the bacterium between pigs, the contamination of mandibular lymph nodes and the contamination of the environment without faecal carriage. Significance and Impact of the Study: The minimal inoculated dose via nasal route to observe transmission of MRSA ST398 between pigs is equal or lower to 2 × 10⁴ colony‐forming units per animal, and faecal excretion seems not to be a necessary condition for horizontal transmission.     

Detection and Quantification of Geobacter lovleyi Strain SZ: Implications for Bioremediation at Tetrachloroethene- and Uranium-Impacted Sites

Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 × 10⁶ and 1 × 10⁴ 16S rRNA gene copies per μl of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per μl of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 × 10⁷ ± 0.1 × 10⁷ per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.     

Improved detection of <Emphasis Type="Italic">Rhodococcus coprophilus</Emphasis> with a new quantitative PCR assay

Agricultural practices, such as spreading liquid manure or the utilisation of land as animal pastures, can result in faecal contamination of water resources. Rhodococcus coprophilus is used in microbial source tracking to indicate animal faecal contamination in water. Methods previously described for detecting of R. coprophilus in water were neither sensitive nor specific. Therefore, the aim of this study was to design and validate a new quantitative polymerase chain reaction (qPCR) to improve the detection of R. coprophilus in water. The new PCR assay was based on the R. coprophilus 16S rRNA gene. The validation showed that the new approach was specific and sensitive for deoxyribunucleic acid from target host species. Compared with other PCR assays tested in this study, the detection limit of the new qPCR was between 1 and 3 log lower. The method, including a filtration step, was further validated and successfully used in a field investigation in Switzerland. Our work demonstrated that the new detection method is sensitive and robust to detect R. coprophilus in surface and spring water. Compared with PCR assays that are available in the literature or to the culture-dependent method, the new molecular approach improves the detection of R. coprophilus.     

Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

Water quality monitoring techniques that target microorganisms in the order Bacteroidales are potential alternatives to conventional methods for detection of fecal indicator bacteria. Bacteroidales and members of the genus Bacteroides have been the focus of microbial source tracking (MST) investigations for discriminating sources of fecal pollution (e.g., human or cattle feces) in environmental waters. For accurate source apportionment to occur, one needs to understand both the abundance of Bacteroides in host feces and the survival of these host-associated microbial markers after deposition in the environment. Studies were undertaken to evaluate the abundance, persistence, and potential for growth of Bacteroidales originating from poultry litter under oxic and anoxic environmental conditions. Bacteroidales abundance, as determined by quantitative PCR (qPCR) with GenBac primers and probe, increased 2 to 5 log gene copies ml⁻¹ and 2 log gene copies g litter⁻¹ under most conditions during incubation of poultry litter in a variety of laboratory microcosm and field mesocosm studies. DNA sequencing of the Bacteroidales organisms in the litter identified taxa with sequences corresponding exactly to the GenBac primer and probe sequences and that were closely related to Bacteroides uniformis, B. ovatus, and B. vulgatus. These results suggest that MST studies using qPCR methods targeting Bacteroidales in watersheds that are affected by poultry litter should be interpreted cautiously. Growth of Bacteroidales originating from poultry litter in environmental waters may occur while Bacteroidales growth from other fecal sources declines, thus confounding the interpretation of MST results.