Discrimination of Viable and Dead Fecal Bacteroidales Bacteria by Quantitative PCR with Propidium Monoazide

Propidium monoazide (PMA) was optimized to discriminate between viable and dead Bacteroides fragilis cells and extracellular DNA at different concentrations of solids using quantitative PCR. Conditions of 100 μM PMA and a 10-min light exposure also excluded DNA from heat-treated cells of nonculturable Bacteroidales in human feces and wastewater influent and effluent.The aim of microbial source tracking (MST) methods is to identify, and in some cases quantify, the dominant sources of fecal contamination in surface waters and groundwater (2, 16). One of the most promising library- and cultivation-independent approaches utilizes fecal Bacteroidales bacteria and quantitative PCR (qPCR) assays to measure gene copies of host-specific genetic markers for 16S rRNA (4, 5, 10, 14). Currently, molecular assays do not directly discriminate between viable and nonviable cells since DNA of both live and dead cells and extracellular DNA can be amplified. Consequently, source tracking data based on detection of genetic markers by PCR cannot distinguish between recent and past contamination events since DNA of selected pathogens can persist after cell death for more than 3 weeks (6). Hence, it would be preferable to detect host-specific markers in viable cells of Bacteroidales bacteria, which are strictly anaerobic microorganisms and unlikely to survive in water.Previous studies have suggested the use of intercalating DNA-binding chemicals combined with PCR to inhibit PCR amplification of DNA derived from dead cells (8, 9, 11, 15). For example, ethidium monoazide (EMA) has been investigated as a means of reducing the PCR signal from DNA originating from dead bacterial cells (7, 15, 19). However, the use of EMA prior to DNA extraction has been found to result in a significant loss of the genomic DNA of viable cells in the case of Escherichia coli 0157:H7, Campylobacter jejuni, and Listeria monocytogenes (3, 7). Recently propidium monoazide (PMA) has been proposed as a more selective agent, penetrating only dead bacterial cells but not viable cells with intact membranes (8). EMA/PMA in combination with PCR or qPCR has been applied to identify viable food-borne pathogens in a simple matrix (3, 7, 8, 11), and possible restrictions in the use of PMA in environmental samples were reported (9, 19). Yet the feasibility of applying PMA in environmental samples or MST studies using fecal Bacteroidales bacteria has not been systematically studied. Any meaningful application of EMA or PMA in stool or natural water samples must consider potential interferences due to particulate matter present in the environmental matrix. Similarly, procedures for the concentration of large volumes of water samples to simultaneously monitor pathogens and MST identifiers can lower the limit of detection (4, 12), but they concentrate solids or other inhibitors of quantitative PCR (qPCR) as well, which might interfere in the covalent binding of PMA to DNA.The objectives of this study were, therefore, the following: (i) to evaluate the applicability of PMA-qPCR methods to detect culturable Bacteroides fragilis, (ii) to determine the feasibility of PMA-qPCR analysis for environmental samples containing different concentrations of solids, and (iii) to validate the utility of the PMA-qPCR method for the detection of fecal Bacteroidales bacteria in defined live and heat-treated mixtures of human feces and in wastewater treatment plant influent and effluent.Pure cultures of Bacteroides fragilis (ATCC 25285) were grown in thioglycolate broth (Anaerobe System, Morgan Hill, CA) under anaerobic conditions in GasPak anaerobic jars (Becton Dickinson Microbiology Systems, Cockeysville, MD). The solids were obtained by hollow-fiber ultrafiltration as described previously (12, 13). Ultrasonification and heat sterilization in an autoclave were used for removing attached bacteria or DNA from solids and inactivating residual DNA. Finally, the solids were resuspended with 1× phosphate-buffered saline (PBS) solution to 100 mg liter⁻¹ or 1,000 mg liter⁻¹ of suspended solids. The concentration of total suspended solids (TSS) was measured using method 2450 C (1). Next, 1 ml of broth medium containing 2 × 10⁹ viable or 2 × 10⁸ heat-treated B. fragilis cells, which had been exposed at 80°C for 20 min, was spiked into 1× PBS buffer solutions containing 0 mg liter⁻¹, 100 mg liter⁻¹, or 1,000 mg liter⁻¹ of TSS. Before the cells were spiked, 1 ml of Bacteroides fragilis cell suspension was enumerated with the Live/Dead BacLight bacterial viability kit (Molecular Probes Inc., Eugene, OR) using a hemacytometer and an Axioskop 2 Plus epifluorescence microscope (Zeiss, Thornwood, NY) equipped with two filter sets (fluorescein isothiocyanate and Texas Red). The inoculated samples were incubated under anaerobic conditions in GasPak anaerobic jars (Becton Dickinson Microbiology Systems, Cockeysville, MD) for 4 h at 20°C to allow sufficient time for the cells to sorb to solids.A fresh human fecal specimen was obtained from a healthy adult. Two grams of feces was suspended in 25 ml 1× PBS. The fecal suspension was diluted 1:10 and 1:100 in a 1× PBS solution, and aliquots were subjected to heat treatment at 80°C for 20 min. The heat-treated fecal portions were mixed with fresh diluted samples (1:10 and 1:100 dilutions) in defined ratios, with fresh feces representing 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% of the total, respectively. Effluent and influent water samples were collected in sterile 2-liter bottles from the University of California, Davis, wastewater treatment plant. The effluent samples were concentrated to approximately 200 ml by hollow-fiber ultrafiltration (12).PMA (Biotium Inc., Hayward, CA) was prepared, stored, and used as described in previous studies (8, 9), but PMA concentrations and light exposure time were varied to determine the optimal condition of PMA-qPCR; the PMA concentrations were 2 μM, 6 μM, 20 μM, and 100 μM. Light exposure times were 1 min, 5 min, 10 min, and 20 min. Genomic DNA was extracted using the FastDNA spin kit for soil (Biomedicals, Solon, OH). Cell lysis was achieved by bead beating using a bead mill Minibread beater (Biospec Products Inc., Bartlesville, OK) at 2,400 rpm for 20 s. Otherwise, DNA extraction was performed according to the manufacturer''s instructions. TaqMan probe and primer assays targeting the rRNA genes of all fecal Bacteroidales bacteria (BacUni-UCD) and mixed human-specific Bacteroidales bacteria (BacHum-UCD), developed by Kildare et al. (4), were used to detect and quantify fecal Bacteroidales bacteria present in fecal and (waste)water samples.We explored the ability of PMA-qPCR to discriminate between viable and heat-killed cells at different solids concentrations using Bacteroides fragilis cultures (Fig. ​(Fig.1).1). PMA did not influence the PCR amplification of DNA derived from viable cells when no solids were present (TSS = 0 mg liter⁻¹) (Fig. ​(Fig.1A).1A). The level of PMA concentration slightly affected the mean cycle threshold differences (ΔC_T) of viable cells at higher solids concentrations (TSS = 100 and 1,000 mg liter⁻¹) (Fig. 1C and E). The signal reductions in the amplification of heat-killed cells were a function of both the PMA concentration and exposure time (Fig. 1B, D, and F). Lower solids concentrations did not inhibit the efficacy of discrimination from heat-killed cells. However, solids at 1,000 mg liter⁻¹ affected the amplification of DNA derived from heat-killed cells. Higher solids concentrations affected the suppression of PCR amplification from heat-treated cells by interfering with the cross-linking of PMA. In agreement with previous reports, the number of viable Bacteroides fragilis cells was underestimated in our study when EMA-treated and untreated samples containing only viable cells were compared because mean ΔC_T values were as high as 10 (data not shown). In contrast to EMA, PMA seems to not penetrate live cells, since higher selectivity of PMA is most probably associated with the higher charge of the molecule (8).Open in a separate windowFIG. 1.Effect of PMA on amplification of BacUni-UCD universal marker in viable and dead Bacteroides fragilis cells with different concentrations of solids. The contour lines represented ΔC_T values and were generated by the Origin Pro 8 software program. The mean cycle threshold differences (ΔC_T) were calculated by subtracting C_T values obtained without PMA treatment from C_T values obtained with PMA treatment. (A and B) ΔC_T for viable cells (A) or dead cells (B) in the absence of added solids. (C and D) ΔC_T for viable cells (C) or dead cells (D) at a solids concentration of 100 mg liter⁻¹. (E and F) ΔC_T for viable cells (E) or dead cells (F) at a solids concentration of 1,000 mg liter⁻¹.A factorial three-way analysis of variance including the PMA concentration, exposure time, and TSS concentration was performed to determine the interferences of solids and the optimal PMA-qPCR condition in the differentiation of viable cells from dead cells (Table ​(Table1).1). The mean ΔC_T of viable cells in the PMA experiments was slightly influenced by the PMA concentration (P = 0.05) in the absence of solids (TSS = 0 mg liter⁻¹), but the effect was biologically insignificant (mean ΔC_T = 0.004). The PMA concentration had a significant effect on ΔC_T values for both viable and dead cells in the presence of higher solids concentrations (TSS = 100 and 1,000 mg liter⁻¹), as shown in Table ​Table1.1. However, the effect of exposure time in PMA treatment was insignificant at a TSS concentration of 1,000 mg liter⁻¹ (P > 0.4). The solids concentration caused significantly different ΔC_T values for viable and dead cells in the PMA treatments (P < 0.001) as determined by factorial three-way analysis. The greatest differences in the mean ΔC_T values between viable and dead cells were seen at 100 μM of PMA and with a 10-min exposure time, as determined by Tukey''s comparison test, for TSS concentrations of 100 mg liter⁻¹ and 1,000 mg liter⁻¹. Ideally, shorter light exposure and a lower concentration of dye can minimize the penetration of live cells. However, these conditions were not compatible with sufficient inhibition of amplification of DNA from dead cells for PMA treatment.

TABLE 1.

Statistical analysis for differences (ΔC_T) between nontreatment and PMA treatment for experiments where Bacteroides fragilis was spiked^a

Specific Detection of Viable Legionella Cells by Combined Use of Photoactivated Ethidium Monoazide and PCR/Real-Time PCR

Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10⁴- to 10⁵-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.     

Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log₁₀ was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.     

Comparative Analysis and Limitations of Ethidium Monoazide and Propidium Monoazide Treatments for the Differentiation of Viable and Nonviable Campylobacter Cells

The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (C_T) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R²) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 μg/ml) and PMA (51.10 μg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10⁴) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.     

Monochloramine Disinfection Kinetics of Nitrosomonas europaea by Propidium Monoazide Quantitative PCR and Live/Dead BacLight Methods

Monochloramine disinfection kinetics were determined for the pure-culture ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) by two culture-independent methods, namely, Live/Dead BacLight (LD) and propidium monoazide quantitative PCR (PMA-qPCR). Both methods were first verified with mixtures of heat-killed (nonviable) and non-heat-killed (viable) cells before a series of batch disinfection experiments with stationary-phase cultures (batch grown for 7 days) at pH 8.0, 25°C, and 5, 10, and 20 mg Cl₂/liter monochloramine. Two data sets were generated based on the viability method used, either (i) LD or (ii) PMA-qPCR. These two data sets were used to estimate kinetic parameters for the delayed Chick-Watson disinfection model through a Bayesian analysis implemented in WinBUGS. This analysis provided parameter estimates of 490 mg Cl₂-min/liter for the lag coefficient (b) and 1.6 × 10⁻³ to 4.0 × 10⁻³ liter/mg Cl₂-min for the Chick-Watson disinfection rate constant (k). While estimates of b were similar for both data sets, the LD data set resulted in a greater k estimate than that obtained with the PMA-qPCR data set, implying that the PMA-qPCR viability measure was more conservative than LD. For N. europaea, the lag phase was not previously reported for culture-independent methods and may have implications for nitrification in drinking water distribution systems. This is the first published application of a PMA-qPCR method for disinfection kinetic model parameter estimation as well as its application to N. europaea or monochloramine. Ultimately, this PMA-qPCR method will allow evaluation of monochloramine disinfection kinetics for mixed-culture bacteria in drinking water distribution systems.As a result of stage 1 and stage 2 disinfectant and disinfection by-product rules, chloramination for secondary disinfection in the United States is predicted to increase to 57% of all surface and 7% of all groundwater treatment systems (49). A recent survey reported that 30% of the respondents currently chloraminate to maintain distribution system residual, and other recent surveys suggest that between 8 and 12% of drinking water utilities are contemplating a future switch to chloramination (3, 43).Although chloramines are considered weaker disinfectants than chlorine for suspended bacteria, chloramines are perceived as more effective disinfectants for a biofilm (25, 53). As a result of their lower reactivity, chloramines are believed to penetrate a biofilm further and thereby to more effectively disinfect biofilm bacteria with depth than chlorine (53).Chloramination comes with the risk of distribution system nitrification (2, 21, 22). Based on utility surveys, 30 to 63% of utilities practicing chloramination for secondary disinfection experience nitrification episodes (3, 21, 43, 54). Nitrification in drinking water distribution systems is undesirable and may result in water quality degradation (e.g., disinfectant depletion, coliform occurrences, or nitrite/nitrate formation) and subsequent noncompliance with existing regulations (e.g., surface water treatment rule or total coliform rule) (2). Thus, nitrification control is a major issue in practice and is likely to become increasingly important as chloramination increases.Unfortunately, our understanding of distribution system nitrification and its control is incomplete, which has made this a topic of considerable ongoing research. Recently, Fleming et al. (12) proposed nitrification potential curves as a possible strategy to prevent nitrification in chloraminated drinking water distribution systems. Use of this concept or other modeling approaches inherently requires knowledge of both the growth and disinfection kinetic parameters of nitrifiers, specifically ammonia-oxidizing bacteria (AOB), inhabiting the distribution system.Several chloramine disinfection studies have been reported for nitrifier cultures (2). However, only one study contains a detailed determination of chloramine disinfection kinetics, having investigated the pure-culture AOB Nitrosomonas europaea (33). In contrast to this pure-culture study, AOB are present as mixed cultures in chloraminated drinking water distribution systems, with Nitrosomonas oligotropha rather than N. europaea representing the dominant AOB found (33, 37, 38). Therefore, determination of disinfection kinetics of mixed-culture AOB likely present in chloraminated drinking water (i.e., N. oligotropha) represents a significant knowledge gap in our understanding of nitrification episodes.Disinfection kinetic parameter determination inherently depends on the method used to quantify viable bacteria. In general, there are two classes of viability determinations, i.e., (i) culture-dependent and (ii) culture-independent methods (5, 16, 27). Culture-dependent methods rely on bacterial growth and include plate counts and most-probable-number (MPN) techniques. Culture-independent methods include activity measures (e.g., substrate uptake or oxygen utilization) and other methods that rely on cell membrane integrity as a viability measure. In general, culture-dependent methods result in faster disinfection kinetics than culture-independent methods.As a first step toward gaining more information on AOB disinfection in chloraminated drinking water distribution systems, a culture-independent method with future applicability to mixed-culture AOB was implemented. In the current research, N. europaea was used. Even though N. europaea has not been found to be the dominant AOB in chloraminated systems, its use in the current research provides a comparison to existing literature. The culture-independent method combines the use of propidium monoazide (PMA), which selectively removes DNA from membrane-compromised cells and/or inhibits its amplification by PCR (29-31), with a quantitative PCR (qPCR) method developed for detection of AOB in chloraminated drinking water distribution systems (36). The results using PMA-qPCR were compared with those obtained using another culture-independent membrane integrity-based technique, the Live/Dead BacLight (LD) method. Furthermore, the experimental conditions were selected (pH 8.0 and a chlorine-to-nitrogen mass ratio of 4:1) such that monochloramine was the dominant chloramine species present, and the results are reported as monochloramine disinfection kinetics. The magnitude of the reported disinfection kinetics was closely related to the respective method used for viability determination. For example, in this research a cell was considered viable or nonviable based on the ability of propidium iodide (PI) or PMA to penetrate its membrane and on subsequent processing according to the respective method.LD was previously used to determine detailed N. europaea disinfection kinetics (33) and provides a baseline comparison for the current research. Oldenburg et al. (33) provided a comparison of estimated disinfection kinetic parameters, using both the culture-dependent AOB MPN technique and LD as viability measures. The estimated disinfection kinetic parameters based on the AOB MPN method were 3 orders of magnitude greater than those obtained with the culture-independent LD method, and the lower disinfection kinetics based on LD were more consistent with AOB persistence in chloraminated drinking water distribution systems. Based on this previous research and because the AOB MPN method requires an incubation period of 21 to 30 days, it was not evaluated in the current research (2).Initially, control experiments were conducted with various proportions of heat-killed cells to verify that both the PMA-qPCR and LD methods detected only viable cells. After the control experiments, a series of batch disinfection experiments were conducted where both PMA-qPCR and LD were utilized to quantify viable bacteria, providing two data sets for disinfection kinetic parameter estimation. Ultimately, the PMA-qPCR method used in this research will be applied to mixed-culture AOB typically present in drinking water distribution systems (i.e., N. oligotropha) (36-38).     

Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide

The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into “dead” cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.     

Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 10² CFU/cm².     

Rapid Quantification of Viable Campylobacter Bacteria on Chicken Carcasses,Using Real-Time PCR and Propidium Monoazide Treatment,as a Tool for Quantitative Risk Assessment

A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method''s performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (C_T) values (R² = 0.993), with a quantification range of 1 × 10² to 1 × 10⁷ CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R² = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment.As Campylobacter remains the leading cause of food-borne bacterial gastrointestinal disease in large parts of the developed world (34), much effort is devoted to improving the detection and elimination of the pathogen, especially in poultry. The ultimate goal is to supply consumers with fresh, Campylobacter-free poultry products, but in order to achieve that goal, it is important to gain more insight into the epidemiology of Campylobacter, to make quantitative risk assessments, and to improve control and intervention strategies.Traditional culture-based detection of Campylobacter bacteria, including enrichment, isolation, and confirmation, is a time-consuming procedure requiring 5 to 6 working days (4, 14). Furthermore, bacterial cells may enter a viable but nonculturable (VBNC) state in which they may have the potential to cause human infection (37) but are not detected by the culture method. The introduction of real-time quantitative PCR (Q-PCR) has enabled faster, more sensitive, and less labor-intensive quantitative detection. Q-PCR methods for food-borne Campylobacter jejuni and C. coli in poultry, which is recognized as an important source of human Campylobacter infections, have been published (11, 12, 15, 38, 46). However, since control strategies mostly focus on reduction of the number of bacterial cells on the chicken carcass, the usefulness of these Q-PCR methods for risk assessment could be limited, since they detect all of the Campylobacter bacteria present in a sample, including the dead cells.The Q-PCR method described in the present study quantifies the three major food-borne Campylobacter species (C. jejuni, C. coli, and C. lari), thereby covering all possible prevalence shifts and coinfections. The PCR assay was previously validated according to the Nordic Organization for Validation of Alternative Microbiological Methods (NordVal) and is certified for detection of Campylobacter bacteria in chickens, cloacal swabs, and boot swabs (7). The present study concerns its suitability for the quantification of Campylobacter bacteria in chicken carcass rinse. Furthermore, a propidium monoazide (PMA) sample treatment step has been incorporated into the method (PMA-PCR), ensuring the quantification of only viable cells with intact membranes. PMA can intercalate into the double-helical DNA available from dead cells with compromised membranes, and upon extensive visible light exposure, cross-linking of the two strands of DNA occurs, leaving it unavailable for PCR amplification (30). PMA is a chemical alteration (additional azide group) of propidium iodide (PI), one of the most frequently applied non-membrane-permeating dyes in flow cytometry, and it can be expected to have the same permeating potential as PI (29). This could be of value from a food safety perspective, since PI penetrates only permeabilized cells and not cells with intact membranes (including the Campylobacter VBNC state), which can still cause infection. Nocker et al. demonstrated that no uptake of PMA occurred in bacterial cells with intact membranes, and PMA was exclusively found in bacteria with compromised membranes (31).PMA sample treatment combined with real-time PCR for detection of viable pathogens has been tested successfully on Listeria monocytogenes and Escherichia coli O157:H7 (31, 36). However, these studies were limited to laboratory-cultured strains and the methods have not been validated on naturally infected samples with the pathogen embedded in a food matrix.This is the first study to establish a correlation between results obtained by PMA-PCR and culture-based enumeration of Campylobacter bacteria for a large number of naturally infected chickens.     

Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology

One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.     

Rapid Quantitative Detection of Listeria monocytogenes in Meat Products by Real-Time PCR

We describe a quick and simple method for the quantitative detection of Listeria monocytogenes in meat products. This method is based on filtration, Chelex-100-based DNA purification, and real-time PCR. It can detect as few as 100 CFU/g and quantify as few as 1,000 CFU/g, with excellent accuracy compared to that of the plate count method. Therefore, it is a promising alternative for the detection of L. monocytogenes in meat products.     

Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S.cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 × 10⁵ to 3.8 CFU/ml in sweet wine and from 5 × 10⁶ to 50CFU/ml in red wine.     

Enumeration of Listeria monocytogenes in raw milk

Enumeration of Listeria monocytogenes in raw milk was compared by direct plating and Most Probable Number (MPN) techniques involving both direct and two-stage enrichments. Direct plating was found to lack sensitivity for the enumeration of low numbers of listerias found in milk but an MPN technique with direct selective enrichment was found to be more suitable than one with two-stage enrichment.     

Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms'' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions.     

PCR技术检测单核细胞增生李斯特氏菌研究进展

单核细胞增生李斯特氏菌(Listeria monocytogenes)是危害公共卫生和食品行业的一种重要人畜共患病致病菌.PCR技术是近年来被广泛地运用到食源性致病菌快速检测的分子生物学方法之一.就PCR技术检测单核细胞增生李斯特氏菌中的特异性鉴别基因、模板DNA制备等关键因素及多重PCR、巢式PCR、反转录PCR与定量PCR等主要技术进行了综述.     

Quantitative detection of Listeria monocytogenes in biofilms by real-time PCR

A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 x 10(2) CFU/cm2.     

Comparison and Characterization of Microbial Communities in Sulfide-rich Wastewater with and without Propidium Monoazide Treatment

A 16S rRNA gene-based culture-independent approach was used to study the bacterial and archaeal communities in a sulfide-rich wastewater. Propidium Monoazide (PMA) treatment was applied to limit the analysis to the fraction of viable cells in environment. A total of 104 and 68 clones respective from bacterial clone library and archaeal library were picked and analyzed by restriction fragment length polymorphism (RFLP). 35 RFLP patterns from bacterial clone library and 10 RFLP patterns from archaeal clone library were unique and the respective clones were selected for sequencing. BLAST analysis and RFLP analysis showed that the bacterial clone library mainly consisted of Gammaproteobacteria (73%), Anaerolineae (6%), Bacilli (5%), Deltaproteobacteria (7%), Clostridia (4%), Bacteroidetes (1%), and Chlorobia (1%); Methanomicrobia (99%) and Thermococci (1%) were the only two lineages of the archaeal domains. This study gave a first insight into the overall microbial structure in a cloth printing and dyeing wastewater treatment plant with high concentration of sulfide and increased knowledge on the applicability of the PMA treatment in combination with PCR-based molecular techniques to analyze only viable cells in microbial ecology.