Propidium Monoazide qPCR Research Articles

Elucidating the Mode of Action of Hybrid Nanoparticles of Cu/Zn Against Copper-Tolerant Xanthomonas euvesicatoria.

The widespread presence of tolerance to copper in Xanthomonas species has resulted in the need to develop alternative approaches to control plant diseases caused by xanthomonads. In recent years, nanotechnological approaches have resulted in the identification of novel materials to control plant pathogens. With many metal-based nanomaterials having shown promise for disease control, an important question relates to the mode of action of these new materials. In this study, we used several approaches, such as scanning electron microscopy, propidium monoazide quantitative polymerase chain reaction, epifluorescence microscopy, and RNA sequencing to elucidate the mode of action of a Cu/Zn hybrid nanoparticle against copper-tolerant strains of Xanthomonas euvesicatoria. We demonstrate that Cu/Zn did not activate copper resistance genes (i.e., copA and copB) in the copper-tolerant bacterium but functioned by disrupting the bacterial cell structure and perturbing important biological processes such as cell respiration and chemical homeostasis.

Detection of Viable Streptococcus equi equi Using Propidium Monoazide Polymerase Chain Reaction.

There is debate around the clinical significance of Streptococcus equi subsp. equi detection in low numbers using quantitative real-time PCR (qPCR). Propidium monoazide (PMA) qPCR has been used to differentiate DNA from viable and nonviable bacterial cells. The aim of this study was to evaluate the ability of PMA eqbE SEQ2190 triplex qPCR to differentiate DNA from viable and nonviable S. equi in positive and suspect positive clinical specimens. 57 stored (frozen and refrigerated) positive (36) or suspect positive (21) clinical specimens (determined via SeeI qPCR as the gold standard) were tested using eqbE SEQ2190 triplex qPCR with (+) and without (-) PMA pre-treatment. Cycle Thresholds (CT) were higher when using PMA indicating a mixture of heat killed (HK) and viable cells. Number of S. equi positive specimens were as follows: 6/57 eqbE +PMA, 13/57 eqbE -PMA (Chi- squared 3.1, p = 0.079); 10/57 SEQ2190 +PMA, 53/57 SEQ2190 -PMA (Chi- squared 65.6, p<0.0001). The mean CT were as follows: 23.88 eqbE -PMA, 29.89 eqbE + PMA (p=0.04); 24.9 SEQ2190 -PMA, 31.9 SEQ2190 +PMA (p<0.0001). PMA qPCR can be used to determine S. equi viability, but testing should be performed on fresh specimens.

To close or open the tank input water valve: Secondary water-supply systems with double tanks will induce a higher microbial risk

Secondary water supply systems (SWSSs) are widely used to supply water to high-rise households in urban residential buildings. A special mode of double tanks with one used while another was spared was noted in SWSSs, which would facilitate microbial growth due to longer water stagnation in the spare tank. There are limited studies on the microbial risk of water samples in such SWSSs. In this study, the input water valves of the operational SWSSs consisting of double tanks were artificially closed and opened on time. Propidium monoazide-qPCR and high-throughput sequencing were performed to systematically investigate the microbial risks in water samples. After closing the tank input water valve, it may take several weeks to replace the bulk water in the spare tank. The residual chlorine concentration in the spare tank decreased by up to 85 % within 2–3 days compared with that in the input water. The microbial communities in the spare and used tank water samples clustered separately. High bacterial 16S rRNA gene abundance and pathogens-like sequences were detected in the spare tanks. Most antibiotic-resistant genes (11/15) in the spare tanks showed an increase in their relative abundance. Moreover, when both tanks within one SWSS were in use, the water quality of the used tank water samples deteriorated to varying degrees. Overall, running SWSSs with double tanks will reduce the replacement rate of water in one storage tank, and consumers who use taps served by the presented SWSSs may have a higher microbial risk.

Detection of suid herpesvirus 1 infectivity in pigs by propidium monoazide-qPCR.

At present, there is no effective experimental method for detecting whether the suid herpesvirus 1 (SHV-1) detected in pigs is infectious. Although the technique of quantitative polymerase chain reaction (qPCR) has significantly improved the detection rate and accuracy of the disease, it does not differentiate between infective and non-infective status of the virus. Propidium monoazide (PMA) is a dye that can be combined with DNA molecules. The decomposition of PMA produces an azene compound covalently crosslinked with DNA molecules, thereby inhibiting PCR amplification of DNA. In this study, the combination of PMA and qPCR was used to determine the infectivity of SHV-1. We optimized the method from the selection of primers, the working concentration of PMA, and the method of inactivation using UV or heat inactivation. We found that when specific primer 1 was used and a PMA working concentration was 50-100 μM, heat inactivation was able to distinguish whether SHV-1 was infectious or not. We also showed that UV prevented the virus from replicating, it did not destroy the capsid of the virus, and therefore, PMA cannot enter the virus and bind to the nucleic acid of the virus. Consequently, there is no way to identify the infectivity of the virus using UV inactivation. The study showed that the method was stable and the detection rate reached 96%. In conclusion, this method exhibited strong specificity and high sensitivity and can identify the infectivity of SHV-1. This method has practical significance for clinical virus isolation and the effects of disinfection of farms.

Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the Gardnerella-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis.

Quantification of the number of living cells in biofilm or after eradication treatments of biofilm, is problematic for different reasons. We assessed the performance of pre-treatment of DNA, planktonic cells and ex vivo vaginal biofilms of Gardnerella with propidium monoazide (PMAxx) to prevent qPCR-based amplification of DNA from killed cells (viability-qPCR). Standard PMAxx treatment did not completely inactivate free DNA and did not affect living cells. While culture indicated that killing of planktonic cells by heat or by endolysin was complete, viability-qPCR assessed only log reductions of 1.73 and 0.32, respectively. Therefore, we improved the standard protocol by comparing different (combinations of) parameters, such as concentration of PMAxx, and repetition, duration and incubation conditions of treatment. The optimized PMAxx treatment condition for further experiments consisted of three cycles, each of: 15 min incubation on ice with 50 µM PMAxx, followed by 15 min-long light exposure. This protocol was validated for use in vaginal samples from women with bacterial vaginosis. Up to log2.2 reduction of Gardnerella cells after treatment with PM-477 was documented, despite the complex composition of the samples, which might have hampered the activity of PM-477 as well as the quantification of low loads by viability-qPCR.

Persistence of Legionella in Routinely Disinfected Heater-Cooler Units and Heater Units assessed by Propidium Monoazide qPCR.

Background: Evidence to date indicates that heater–cooler units (HCUs) and heater units (HUs) can generate potentially infectious aerosols containing a range of opportunistic pathogens such as Mycobacterium chimaera, other non-tuberculous mycobacterial (NTM) species, Pseudomonas aeruginosa and Legionella spp. Our purpose was to determine the extent of Legionella contamination and total viable count (TVC) in HCUs and HUs and to analyze the relationship by water system design of devices of two different brands (LivaNova vs. Maquet). Methods: Legionella spp. were detected and quantified by our optimized PMA-qPCR protocol; TVCs were assessed according to ISO protocol 6222. Analyses were performed in the first sampling round and after six months of surveillance. Results: Overall, Legionella spp. was detected in 65.7% of devices. In the second sampling round, Legionella positivity rates were significantly lower in water samples from the Maquet devices compared to the LivaNova ones (27.3% vs. 61.5%). LivaNova HCUs also yielded more Legionella, and aquatic bacteria counts than Maquet in both first and second-round samples. Conclusions: We recommend that all surgical patients and staff exposed to aerosols from thermoregulatory devices should be followed up for Legionella infection and that microbiological surveillance on such devices should be conducted regularly as precautionary principle.

Behavior of Mycobacterium avium paratuberculosis in Lighvan cheese tracked by propidium monoazide qPCR and culture

Mycobacterium avium subsp. Paratuberculosis (Map) is an important animal pathogen with a worldwide distribution that may contribute to Crohn's disease in humans. This study aimed to investigate the behavior of Map in Lighvan cheese with special reference to the strains of Map, inoculum load, and storage time. One laboratory and two native strains of Map were inoculated (102 and 103Map cell/mL) to cheese milk. The behavior of Map throughout the manufacture, ripening, and storage stage of Lighvan cheese tracked using propidium monoazide (PMA) qPCR and culture examination. According to the results, PMA-qPCR showed parallel outcomes in comparison with the culture assay. Besides, in all cheese batches Map was not affected during the ripening; however, throughout the storage period Map population decreased depending on the strain of Map. Trend analysis revealed no significant difference between the persistence of sheep and goat strains, but in all cheese batches, these native strains persisted significantly (p < 0.05) longer than the laboratory strain. In all treatments, the decreasing rate of Map found independent of the initial inoculum load. Nonetheless, in the cheese batches with higher initial inoculum, Map persisted two months longer.

Comparison of the performance of the biofilm sampling methods (swab, sponge, contact agar) in the recovery of Listeria monocytogenes populations considering the seafood environment conditions

AimsThe aim of this study was to evaluate the performance of sampling methods [contact plates, sponges, and swabs] in the recovery of biofilm Listeria monocytogenes populations considering the seafood environment conditions (nature of conditioning, of materials and bacterial species). Methods and resultsDifferent materials (stainless steel, polyvinyl chloride, polyurethane) were conditioned with two fish filtrates, the ready-to-eat the most consumed in Europe (smoked salmon, cod). After, we added the suspension of Listeria monocytogenes, alone or with Pseudomonas fluorescens or Carnobacterium strains, and incubated for 48 h at 8 °C. Then, the 48 h-biofilms were sampled with different methods (contact plates, sponges, and swabs). The cultivable bacterial populations were enumerated on agar, while the L. monocytogenes total and viable populations were quantified by qPCR and propidium monoazide-qPCR (PMA-qPCR), respectively. The amount of L. monocytogenes in biofilms was affected only by the nature of the conditioning with lowest adherent bacteria with cod versus with smoked salmon conditioning. Considering the amount of total population, the swab displayed the lowest values versus the sponges and the contact plates. An explanation was that the observations of the swab by microscopy showed the bacteria trapped within it. The recovery of cultivable bacterial populations was not significantly different with the three sampling methods. On the contrary, we showed that the VBNC populations were only detached by two of three methods (contact plates, sponges) while for the dead populations, those were contact plates and swabs. ConclusionsThe nature of the conditioning influenced the amount of the bacteria in biofilms. And the performance of the recovery of the bacterial populations (dead, VBNC, cultivable) was dependent on the methods used. Significance and impact of the studyThis study showed that the seafood environmental conditions influenced the biofilm formation and the assessment of the efficiency of cleaning and disinfectant operations could be significantly affected by the used sampling methods.

Colonization of Dental Unit Waterlines by Helicobacter pylori: Risk of Exposure in Dental Practices.

Dental unit waterlines (DUWLs) can be considered one of the possible routes of H. pylori transmission, although its presence in DUWLs has not yet been investigated thoroughly. The present study aimed to discover the prevalence of H. pylori and oral streptococci (S. oralis and S. mutans) in DUWLs to evaluate the risk of exposure to human pathogens in dental practices. We collected the output water from 60 dental chair units (DCUs) in 26 private dentistry settings in Turin, searching for H. pylori and oral streptococci (OS) DNA, with a polymerase chain reaction (PCR) technique. At the same time, dentists completed a questionnaire about their DCUs, their main activities, the presence of anti-retraction devices, their attitudes about disinfection, etc. No dental chair unit tested was contaminated with H. pylori or S. mutans; only one dental chair was contaminated with S. oralis (1.7%). Considering the results, we can state that: (i) the lack of H. pylori DNA in water samples analyzed, suggests that municipal water is presumably treated with a sufficient chlorine level to inactivate DNA over time; (ii) the aspiration of oral fluids is limited by anti-retraction valves fitted distally to hand pieces; (iii) propidium monoazide qPCR (PMA-qPCR) could be a good technique to investigate and monitor potential environmental sources of infections such as DUWLs.

Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold-starvation using propidium monoazide real-time polymerase chain reaction.

Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation-based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO9 and propidium iodide (PI) staining and real-time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15-16·69. During cold-starvation, PMA qPCR selectively excluded DNAs from heat-killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold-starvation. Currently, VBNC cells are routinely detected by SYTO9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real-time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold-starvation conditions.

Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk.

Streptococcus agalactiae is an important pathogen causing bovine mastitis. The aim of this study was to develop a simple and specific method for direct detection of S. agalactiae from milk products. Propidium monoazide (PMA) and sodium dodecyl sulfate (SDS) were utilized to eliminate the interference of dead and injured cells in qPCR. Lysozyme (LYZ) was adopted to increase the extraction efficiency of target bacteria DNA in milk matrix. The specific primers were designed based on cfb gene of S. agalactiae for qPCR. The inclusivity and exclusivity of the assay were evaluated using 30 strains. The method was further determined by the detection of S. agalactiae in spiked milk. Results showed significant differences between the SDS–PMA–qPCR, PMA–qPCR and qPCR when a final concentration of 10 mg/ml (R2 = 0.9996, E = 95%) of LYZ was added in DNA extraction. Viable S. agalactiae was effectively detected when SDS and PMA concentrations were 20 μg/ml and 10 μM, respectively, and it was specific and more sensitive than qPCR and PMA–qPCR. Moreover, the SDS–PMA–qPCR assay coupled with LYZ was used to detect viable S. agalactiae in spiked milk, with a limit of detection of 3 × 103 cfu/ml. Therefore, the SDS–PMA–qPCR assay had excellent sensitivity and specificity for detection of viable S. agalactiae in milk.

Evaluation of propidium monoazide and long-amplicon qPCR as an infectivity assay for coliphage

Standardized and rapid assays for viable viral pathogens are needed to inform human health risk assessments. Conventional qPCR is designed to enumerate the gene copies of an organism in a sample, but does not identify those that originated from a viable pathogen. This study was undertaken to evaluate modified qPCR methods as infectivity assays for the enumeration of infectious MS2 coliphage. Propidium monoazide (PMA) treatment coupled with long-amplicon qPCR assays were assessed for their ability to quantify infectious MS2 in pure cultures and following inactivation by a range of UV light exposures and chlorine doses. The qPCR results were compared to the plaque assay, which was used as the standard to indicate the level of infectious MS2 in each sample. For pure cultures, PMA-qPCR results were not significantly different from the plaque assay (p>0.05). At >4 log inactivation, combined PMA and long-amplicon qPCR assays overestimated the level of infectious MS2 remaining (p<0.05). The most accurate long-amplicon qPCR infectivity assay targeted a 624-bp region at the 5′ end of the genome. Modified qPCR approaches may be useful tools to monitor the loss of infectivity as a result of disinfection processes.

In vitro gastrointestinal resistance of Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 in soy and/or milk-based synbiotic apple ice creams

The viability and resistance to simulated gastrointestinal (GI) conditions of Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 in synbiotic ice creams, in which milk was replaced by soy extract and/or whey protein isolate (WPI) with inulin, were investigated. The ice creams were showed to be satisfactory vehicles for La-5 and Bb-12 (populations around 7.5logCFU/g), even after the whole storage period (84days/−18°C). In all formulations, the propidium monoazide qPCR (PMA-qPCR) analysis demonstrated that probiotics could resist the in vitro GI assay, with significant survival levels, achieving survival rates exceeding 50%. Additionally, scanning electron microscopy images evidenced cells with morphological differences, suggesting physiological changes in response to the induced stress during the in vitro assay. Although all formulations provided resistance to the probiotic strains under GI stress, the variation found in probiotic survival suggests that GI tolerance is indeed affected by the choice of the food matrix.

Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production

The microbial requirements defined in many quality assurance guidelines and standards of primary production demand the establishment of microbial sampling programs. Recently, a q-PCR Escherichia coli assay has been reported as a good method to quantify the presence of fecal indicator bacterial in groundwater samples. This study focuses on the optimization, validation and application of a qPCR method combined with propidium monoazide (PMA) treatment to exclude DNA from dead cells. A first screening consisting of six primer sets targeting single and multi-copy of E. coli were tested to evaluate the sensitivity of the assay. After that, four primer sets were selected, combined with PMA treatment and their capacity to distinguish viable cells when combined with a background of dead cells was assessed. A primer set targeting the 23S rRNA gene was 10-fold more sensitive than the rest of primers, enabling the detection of low concentrations of viable E. coli cells. This assay also exhibited good repeatability and reproducibility, which indicates the robustness of the method. Optimized and validated PMA-qPCR was used to enumerate E. coli in environmental samples including irrigation water and fresh produce. The results were compared with the levels quantified using qPCR and cultivation-based techniques. Counts of E. coli using plate count assay were significantly lower than the levels obtained by molecular techniques (PMA-qPCR and qPCR) in both irrigation water and fresh produce. E. coli PMA-qPCR enumeration method showed similar results as qPCR quantification, although the PMA-qPCR treatment seemed to a good alternative to distinguish between viable and dead cells. It can be concluded that the optimized PMA-qPCR assay can be used by the industry in microbial sampling programs, helping them with the implementation of Good Agricultural Practices (GAP).

Quantification of Pseudomonas aeruginosa in multispecies biofilms using PMA-qPCR.

Multispecies biofilms are an important healthcare problem and may lead to persistent infections. These infections are difficult to treat, as cells in a biofilm are highly resistant to antimicrobial agents. While increasingly being recognized as important, the properties of multispecies biofilms remain poorly studied. In order to do so, the quantification of the individual species is needed. The current cultivation-based approaches can lead to an underestimation of the actual cell number and are time-consuming. In the present study we set up a culture-independent approach based on propidium monoazide qPCR (PMA-qPCR) to quantify Pseudomonas aeruginosa in a multispecies biofilm. As a proof of concept, we explored the influence of the combined presence of Staphylococcus aureus, Streptococcus anginosus and Burkholderia cenocepacia on the antimicrobial susceptibility of P. aeruginosa using this PMA-qPCR approach.

UV disinfection induces a VBNC state in Escherichia coli and Pseudomonas aeruginosa.

The occurrence of a viable but nonculturable (VBNC) state in bacteria may dramatically underestimate the health risks associated with drinking water. Therefore, the potential for UV treatment to induce a VBNC state in Escherichia coli and Pseudomonas aeruginosa was investigated. UV disinfection effectively reduced the culturability of E. coli and P. aeruginosa, with the destruction of nucleic acids demonstrated using gadA long gene fragment qPCR amplification. Following UV radiation, copy numbers for the high transcriptional levels of the 16S rRNA gene varied insignificantly in both strains, confirming results from plate counting assays indicating that VBNC states were induced in both strains. Furthermore, the virulence genes gadA and oprL remained highly expressed, suggesting that the VBNC bacteria still displayed pathogenicity. Propidium monoazide qPCR indicated that cell membranes remained intact even at a UV dose of 300 mJ/cm(2). The RT-qPCR results after UV and chlorine treatments in E. coli were significantly different (8.41 and 5.59 log units, respectively), further confirming the induction of VBNC bacteria induced by UV radiation. Finally, resuscitation was achieved, with E. coli showing greater resuscitation ability than P. aeruginosa. These results systematically revealed the potential health risks of UV disinfection and strongly suggest a combined disinfection strategy.

Repeated cycles of chemical and physical disinfection and their influence on Mycobacterium avium subsp. paratuberculosis viability measured by propidium monoazide F57 quantitative real time PCR

Mycobacterium avium subsp. paratuberculosis (MAP) has a high degree of resistance to chemical and physical procedures frequently used for the elimination of other bacteria. Recently, a method for the determination of viability by exposure of MAP to propidium monoazide (PMA) and subsequent real time quantitative PCR (qPCR) was established and found to be comparable with culture. The aim of this study was to apply the PMA qPCR method to determine the impact of increasing concentration or time and repeated cycles of the application of selected disinfectants on MAP viability. Different MAP isolates responded to the same type of stress in different ways. The laboratory strain CAPM 6381 had the highest tolerance, while the 8819 low-passage field isolate was the most sensitive. Ultraviolet exposure caused only a partial reduction in MAP viability; all MAP isolates were relatively resistant to chlorine. Only the application of peracetic acid led to the total elimination of MAP. Repeated application of the treatments resulted in more significant decreases in MAP viability compared to single increases in the concentration or time of exposure to the disinfectant.

Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide

Potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, are obligate parasites of solanaceous plants, causing severe losses in several potato growing areas throughout the world. To date, management of PCN is related to nematode population densities estimated as eggs per gram of soil, without considering the actual number of viable juveniles within the cysts. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB‐treated nematodes. Although MB is considered to be reliable in staining embryonated juveniles within eggs and cysts, it is a time‐ and labour‐consuming assay. In the present work, a real‐time PCR (qPCR)‐based method combined with propidium monoazide (PMA), a photoreactive DNA‐intercalating dye, was developed for the quantification of viable PCN. This dye renders exposed DNA of dead cells unable to be amplified by PCR, and thus only DNA from viable/intact PCN juveniles is amplified and detected. The novelty of the present method lies in the simultaneous quantitative and qualitative estimation of viable PCN inocula using species‐specific primers and TaqMan probes. The PMA–qPCR viability method (v‐PCR) developed for the two Globodera species successfully discriminated dead from living specimens in heat‐treated samples and eggs in old and newly formed cysts. Interestingly, the detection of DNA from 34‐year‐old nematode cysts stored at room temperature was observed. In conclusion, the proposed v‐PCR method should prove to be very useful for the routine determination of PCN viability from field samples.

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.

Assessment of viable periodontal pathogens by reverse transcription quantitative polymerase chain reaction

Molecular biological methods for the detection of periodontitis-associated bacteria based on DNA amplification have many advantages over classical culture techniques. However, when it comes to assessing immediate therapeutic success, e.g. reduction of viable bacteria, DNA-based polymerase chain reaction is unsuitable because it does not distinguish between live and dead bacteria. Our objective was to establish a simple RNA-based method that is easily set up and allows reliable assessment of the live bacterial load. We compared conventional quantitative real-time PCR (qPCR), propidium monoazide-qPCR and reverse transcription qPCR (RT-qPCR) for the detection of periodontal pathogens after antibiotic treatment in vitro. Applicability was tested using clinical samples of subgingival plaque obtained from patients at different treatment stages. The bacterial load was remarkably stable over prolonged periods when assessed by conventional qPCR, while both propidium monoazide intercalation as well as cDNA quantitation showed a decline according to decreasing numbers of viable bacteria after antibiotic treatment. Clinical samples of subgingival plaque were directly subjected to DNase I treatment and RT without previous extraction or purification steps. While the results of the DNA- and RNA-based methods are comparable in untreated patients, the classical qPCR frequently detected substantial bacterial load in treated patients where RT-qPCR no longer indicates the presence of those pathogens. The disagreement rates ranged between 4 and 20% in first visit patients and 8-50% in the group of currently treated patients. We propose to use RNA-based detection methods to verify the successful eradication of periodontal pathogens.