Viable Campylobacter jejuni on Eggshells and Its Potential to Cross-contaminate Egg White and Yolk When Using a Manual Separation Technique, Determined by Culture and Propidium Monoazide (PMA) qPCR.

Manual separation of egg yolk from egg white using the eggshell is common practice in private households. For this, the egg is cracked and both components are separated by passing the egg yolk back and forth between the two halves of the eggshell, allowing the egg white to drip down while the egg yolk remains in the shell. During this process, the egg content naturally gets in contact with the outside of the eggshell, which might lead to a cross-contamination with its microorganisms, thus was correspondingly assessed in this study. Campylobacter jejuni is one of the most important zoonotic pathogens that can be found on eggshells. Therefore, this bacterium was used to artificially contaminate the eggshells (n = 22) with concentrations of 3.1 ± 0.6 log10 cfu/g. After separating the egg yolk from the egg white, cross-contamination was determined using culture and qPCR. Altogether, cross-contaminations with C. jejuni were found in 15 egg white (68%) and in three egg yolk (14%) samples. Afterward, 90 eggs from 30 egg packs from different producers in and around Munich (Germany) were obtained for field study purposes. To address the problem of culturing due to a possible viable but nonculturable (VBNC) status of C. jejuni, a method to differentiate viable and dead C. jejuni on eggshell using 10 µM propidium monoazide (PMA) and qPCR was developed. As a result, seven egg packs (23%) were positive for C. jejuni. Of these, only one (3%) was contaminated with viable cells, but still in a concentration of 3.3 log10 cells/g shell. According to these results and considering that eggshells might also be naturally contaminated with other pathogens, the authors recommend avoiding the manual separation technique of egg white and yolk by the eggshell. Especially if raw egg white or yolk is used for preparation of not sufficiently heated foods, where contaminating pathogens are not inactivated during processing, this technique might be a safety hazard for the consumer.

On-site filtration of large sample volumes improves the detection of opportunistic pathogens in drinking water distribution systems.

In this study, we compared conventional vacuum filtration of small volumes through disc membranes (effective sample volumes for potable water: 0.3-1.0 L) with filtration of high volumes using ultrafiltration (UF) modules (effective sample volumes for potable water: 10.6-84.5 L) for collecting bacterial biomass from raw, finished, and tap water at seven drinking water systems. Total bacteria, Legionella spp., Legionella pneumophila, Mycobacterium spp., and Mycobacterium avium complex in these samples were enumerated using both conventional quantitative PCR (qPCR) and viability qPCR (using propidium monoazide). In addition, PCR-amplified gene fragments were sequenced for microbial community analysis. The frequency of detection (FOD) of Legionella spp. in finished and tap water samples was much greater using UF modules (83% and 77%, respectively) than disc filters (24% and 33%, respectively). The FODs for Mycobacterium spp. in raw, finished, and tap water samples were also consistently greater using UF modules than disc filters. Furthermore, the number of observed operational taxonomic units and diversity index values for finished and tap water samples were often substantially greater when using UF modules as compared to disc filters. Conventional and viability qPCR yielded similar results, suggesting that membrane-compromised cells represented a minor fraction of total bacterial biomass. In conclusion, our research demonstrates that large-volume filtration using UF modules improved the detection of opportunistic pathogens at the low concentrations typically found in public drinking water systems and that the majority of bacteria in these systems appear to be viable in spite of disinfection with free chlorine and/or chloramine.IMPORTANCEOpportunistic pathogens, such as Legionella pneumophila, are a growing public health concern. In this study, we compared sample collection and enumeration methods on raw, finished, and tap water at seven water systems throughout the State of Minnesota, USA. The results showed that on-site filtration of large water volumes (i.e., 500-1,000 L) using ultrafiltration membrane modules improved the frequency of detection of relatively rare organisms, including opportunistic pathogens, compared to the common approach of filtering about 1 L using disc membranes. Furthermore, results from viability quantitative PCR (qPCR) with propidium monoazide were similar to conventional qPCR, suggesting that membrane-compromised cells represent an insignificant fraction of microorganisms. Results from these ultrafiltration membrane modules should lead to a better understanding of the microbial ecology of drinking water distribution systems and their potential to inoculate premise plumbing systems with opportunistic pathogens where conditions are more favorable for their growth.

Rapid and Accurate Quantification of Viable Lactobacillus Cells in Infant Formula by Flow Cytometry Combined with Propidium Monoazide and Signal-Enhanced Fluorescence In Situ Hybridization.

Lactobacillus is an important member of the probiotic bacterial family for regulating human intestinal microflora and preserving its normalcy, and it has been widely used in infant formula. An appropriate and feasible method to quantify viable Lactobacilli cells is urgently required to evaluate the quality of probiotic-fortified infant formula. This study presents a rapid and accurate method to count viable Lactobacilli cells in infant formula using flow cytometry (FCM). First, Lactobacillus cells were specifically and rapidly stained by oligonucleotide probes based on a signal-enhanced fluorescence in situ hybridization (SEFISH) technique. A DNA-binding fluorescent probe, propidium monoazide (PMA), was then used to accurately recognize viable Lactobacillus cells. The entire process of this newly developed PMA-SEFISH-FCM method was accomplished within 2.5 h, which included pretreatment, dual staining, and FCM analysis; thus, this method showed considerably shorter time-to-results than other rapid methods. This method also demonstrated a good linear correlation (R2 = 0.9994) with the traditional plate-based method with a bacterial recovery rate of 91.24%. To the best of our knowledge, the present study is the first report of FCM combined with PMA and FISH for the specific detection of viable bacterial cells.

Propidium monoazide PCR, a method to determine OsHV-1 undamaged capsids and to estimate virus Lethal Dose 50.

Ostreid herpes virus 1 (OsHV-1) has been classified within the Malacoherpesviridae family from the Herpesvirales order. OsHV-1 is the etiological agent of a contagious viral disease of Pacific oysters, C. gigas, affecting also other bivalve species. Mortality rates reported associated with the viral infection vary considerably between sites and countries and depend on the age of affected stocks. A variant called µVar has been reported since 2008 in Europe and other variants in Australia and in New Zealand last decade. These variants are considered as the main causative agents of mass mortality events affecting C. gigas. Presently there is no established cell line that allows for the detection of infectious OsHV-1. In this context, a technique of propidium monoazide (PMA) PCR was developed in order to quantify "undamaged" capsids. This methodology is of interest to explore the virus infectivity. Being able to quantify viral particles getting an undamaged capsid (not only an amount of viral DNA) in tissue homogenates prepared from infected oysters or in seawater samples can assist in the definition of a Lethal Dose (LD) 50 and gain information in the experiments conducted to reproduce the viral infection. The main objectives of the present study were (i) the development/optimization of a PMA PCR technique for OsHV-1 detection using the best quantity of PMA and verifying its effectiveness through heat treatment, (ii) the definition of the percentage of undamaged capsids in four different tissue homogenates prepared from infected Pacific oysters and (iii) the approach of a LD50 during experimental viral infection assays on the basis of a number of undamaged capsids. Although the developped PMA PCR technique was unable to determine OsHV-1 infectivity in viral supensions, it could greatly improve interpretation of virus positive results obtained by qPCR. This technique is not intended to replace the quantification of viral DNA by qPCR, but it does make it possible to give a form of biological meaning to the detection of this DNA.

Real-time and visual detection of viable Salmonella in milk by a competitive annealing mediated isothermal amplification (CAMP) combined with propidium monoazide (PMA).

Salmonella is a common pathogen in raw milk. The conventional isothermal amplification assay cannot distinguish viable bacteria from dead bacteria, which may cause false positive results or overestimate the number of viable bacteria. This study proposed a competitive annealing mediated isothermal amplification (CAMP) combined with propidium monoazide (PMA) for real-time and visual detection of viable Salmonella in milk. Based on the invA gene, specific CAMP primers were constructed. Moreover, the primers for accelerating the CAMP reaction were also designed and added to the reaction system. The real-time PMA-CAMP showed a LOD of 102 CFU mL-1 for quantitative detection of viable Salmonella in spiked milk samples, and the recovery rate was 80-106%. The visual PMA-CAMP can be performed under isothermal conditions using a portable dry bath, and the positive results can be directly observed by the colorimetric change from violet to sky blue. Without enrichment step, viable Salmonella could be detected with a LOD of 102 CFU mL-1. With enrichment step, even if the initial inoculation level is 1 CFU mL-1, the visual PMA-CAMP could still detect the presence of viable Salmonella in milk samples. Therefore, the developed PMA-CAMP assays are suitable for the monitoring of viable Salmonella contamination in milk.

Rapid Quantification of Infectious Cucumber green mottle mosaic virus in Watermelon Tissues by PMA Coupled with RT-qPCR.

Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus genus and is an important quarantine virus of cucurbit crops. Seedborne transmission is one of the principal modes for CGMMV spread, and effective early detection is helpful to prevent the occurrence of the disease. Quantitative real-time reverse-transcription PCR (RT-qPCR) is a sensitive and rapid method for detecting CGMMV nucleic acids, but it cannot distinguish between infectious and noninfectious viruses. In the present work, a propidium monoazide (PMA) assisted RT-qPCR method (PMA-RT-qPCR) was developed to rapidly distinguish infectious and inactive CGMMV. PMA is a photoactive dye that can selectively react with viral RNA released or inside inactive CGMMV virions but not viral RNA inside active virions. The formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be amplified. The primer pair cp3-1F/cp3-1R was designed based on the coat protein (cp) gene for specific amplification of CGMMV RNA by RT-qPCR. The detection limit of the RT-qPCR assay was 1.57 × 102 copies·µL-1. PMA at 120 µmol·L-1 was suitable for the selective quantification of infectious CGMMV virions. Under optimal conditions, RT-qPCR detection of heat-inactivated CGMMV resulted in Ct value differences larger than 16 between PMA-treated and non-PMA-treated groups, while Ct differences less than 0.23 were observed in the detection of infectious CGMMV. For naturally contaminated watermelon leaf, fruit and seedlot samples, infectious CGMMV were quantified in 13 out of the 22 samples, with infestation levels of 102~105 copies·g-1. Application of this assay enabled the selective detection of infectious CGMMV and facilitated the monitoring of the viral pathogen in watermelon seeds and tissues, which could be useful for avoiding the potential risks of primary inoculum sources.

A new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of marine-derived Bacillus velezensis B-9987.

Marine-derived Bacillus velezensis B-9987 is an important biocontrol bacterium with a broad-spectrum antibacterial effect. The traditional plate counting method is widely used for quantitative detection of viable bacteria and spores but has some disadvantages such as being laborious and time-consuming (at least 24-48 h). This study aimed to develop a new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of B-9987. The specific primers were designed for qPCR amplification based on the conserved region of the bmmA gene (encoding a malonyl CoA-ACP transacylase) of B-9987. According to the characteristic that propidium monoazide (PMA) dye can distinguish viable and dead bacteria, the optimal PMA concentration of 10 µg/ml and optimal exposure time of 10 min were achieved under PMA treatment conditions. The B-9987 spores' genomic DNA was successfully extracted after the spore coat was removed and spore germination was induced. The quantification limits of the PMA-qPCR method were determined for viable B-9987 bacteria, spores in pure culture, and spores in marine Bacillus wettable powder (marine Bacillus WP) and were 1.5 × 103 CFU/ml, 6.5 × 102 CFU/ml, and 103 CFU/ml, respectively. Compared with the qPCR method, the PMA-qPCR method could sensitively detect viable bacteria in the viable/dead bacterial mixture. In this study, the developed PMA-qPCR method was found to have excellent sensitivity and specificity in the context of a pure culture of B-9987 strain, which could accurately and rapidly detect viable B-9987 bacteria within 3-4 h and viable B-9987 spores in marine Bacillus WP within 4-6 h.

Changes in physiological states of Salmonella Typhimurium measured by qPCR with PMA and DyeTox13 Green Azide after pasteurization and UV treatment.

Diarrheal diseases caused by Salmonella pose a major threat to public health, and assessment of bacterial viability is critical in determining the safety of food and drinking water after disinfection. Viability PCR could overcome the limitations of traditional culture-dependent methods for a more accurate assessment of the viability of a microbial sample. In this study, the physiological changes in Salmonella Typhimurium induced by pasteurization and UV treatment were evaluated using a culture-based method, RT-qPCR, and viability PCR. The plate count results showed no culturable S. Typhimurium after the pasteurization and UV treatments, while viability PCR with propidium monoazide (PMA) and DyeTox13-qPCR indicated that the membrane integrity of S. Typhimurium remained intact with no metabolic activity. The RT-qPCR results demonstrated that invasion protein (invA) was detectable in UV-treated cells even though the log2-fold change ranged from - 2.13 to - 5.53 for PMA treatment. However, the catalytic activity gene purE was under the detection limit after UV treatment, indicating that most Salmonella entered metabolically inactive status after UV disinfection. Also, viability PCRs were tested with artificially contaminated eggs to determine physiological status on actual food matrices. DyeTox13-qPCR methods showed that most Salmonella lost their metabolic activity but retained membrane integrity after UV disinfection. RT-qPCR may not determine the physiological status of Salmonella after UV disinfection because mRNA could be detectable in UV-treated cells depending on the choice of target gene. Viability PCR demonstrated potential for rapid and specific detection of pathogens with physiological states such as membrane integrity and metabolic activity.Key Points• Membrane integrity of Salmonella remained intact with no metabolic activity after UV.• mRNA could be detectable in UV-treated cells depending on the choice of target gene.• Viability PCR could rapidly detect specific pathogens with their physiological states.

A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In Vivo.

BACKGROUND: Polymerase chain reaction (PCR) is an important means by which to study the urine microbiome and is emerging as possible alternative to urine cultures to identify pathogens that cause urinary tract infection (UTI). However, PCR is limited by its inability to differentiate DNA originating from viable, metabolically active versus non-viable, inactive bacteria. This drawback has led to concerns that urobiome studies and PCR-based diagnosis of UTI are confounded by the presence of relic DNA from non-viable bacteria in urine. Propidium monoazide (PMA) dye can penetrate cells with compromised cell membranes and covalently bind to DNA, rendering it inaccessible to amplification by PCR. Although PMA has been shown to differentiate between non-viable and viable bacteria in various settings, its effectiveness in urine has not been previously studied. We sought to investigate the ability of PMA to differentiate between viable and non-viable bacteria in urine. METHODS: Varying amounts of viable or non-viable uropathogenic E. coli (UTI89) or buffer control were titrated with mouse urine. The samples were centrifuged to collect urine sediment or not centrifuged. Urine samples were incubated with PMA and DNA cross-linked using blue LED light. DNA was isolated and uidA gene-specific PCR was performed. For in vivo studies, mice were inoculated with UTI89, followed by ciprofloxacin treatment or no treatment. After the completion of ciprofloxacin treatment, an aliquot of urine was plated on non-selective LB agar and another aliquot was treated with PMA and subjected to uidA-specific PCR. RESULTS: PMA's efficiency in excluding DNA signal from non-viable bacteria was significantly higher in bacterial samples in phosphate-buffered saline (PBS, dCT=13.69) versus bacterial samples in unspun urine (dCT=1.58). This discrepancy was diminished by spinning down urine-based bacterial samples to collect sediment and resuspending it in PBS prior to PMA treatment. In 3 of 5 replicate groups of UTI89-infected mice, no bacteria grew in culture; however, there was PCR amplification of E. coli after PMA treatment in 2 of those 3 groups. CONCLUSION: We have successfully developed PMA-based PCR methods for amplifying DNA from live bacteria in urine. Our results suggest that non-PMA bound DNA from live bacteria can be present in urine, even after antibiotic treatment. This indicates that viable but non-culturable E. coli can be present following treatment of UTI, and may explain why some patients have persistent symptoms but negative urine cultures following UTI treatment.

Real-time recombinase-aided amplification with improved propidium monoazide for the rapid detection of viable Escherichia coli O157:H7 in milk.

Escherichia coli O157:H7, the causative agent of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in humans, generates a effective harm to community health because of its high pathogenicity. A real-time recombinase-aided amplification (rRAA) is an emerging method for nucleic acid detection. However, genomic DNA of bacteria could exist in food and the environment for a long time after death and could be amplified by rRAA assay, resulting in false-positive signal; thus, developing a fast and sensitive method is necessary to detect viable foodborne pathogens in food products. In our research, rRAA assay coupled with an enhanced nucleic acid binding dye named improved propidium monoazide (PMAxx) was established and applied in viable E. coli O157:H7 identification in skim milk. The PMAxx could eliminate interference from dead bacteria by permeating impaired membranes and covalently linking to DNA to prevent DNA amplification. The PMAxx-rRAA assay was performed with high sensitivity and good specificity. The PMAxx-rRAA assay could detect as low as 5.4 × 100 cfu/mL of viable E. coli O157:H7 in pure culture, and 7.9 × 100 cfu/mL of viable E. coli O157:H7 in skim milk. In addition, the PMAxx-rRAA assay was performed in the presence of a high concentration of dead bacteria or nontarget bacteria in skim milk to verify the capacity to resist interference from dead bacteria and nontarget bacteria. Therefore, the established PMAxx-rRAA assay is a valuable tool for the identification of viable E. coli O157:H7 in complex food matrix.

The Viable Microbiome of Human Milk Differs from the Metataxonomic Profile.

Bacteria in human milk contribute to the establishment of the infant gut microbiome. As such, numerous studies have characterized the human milk microbiome using DNA sequencing technologies, particularly 16S rRNA gene sequencing. However, such methods are not able to differentiate between DNA from viable and non-viable bacteria. The extent to which bacterial DNA detected in human milk represents living, biologically active cells is therefore unclear. Here, we characterized both the viable bacterial content and the total bacterial DNA content (derived from viable and non-viable cells) of fresh human milk (n = 10). In order to differentiate the living from the dead, a combination of propidium monoazide (PMA) and full-length 16S rRNA gene sequencing was used. Our results demonstrate that the majority of OTUs recovered from fresh human milk samples (67.3%) reflected DNA from non-viable organisms. PMA-treated samples differed significantly in their bacterial composition compared to untreated samples (PERMANOVA p < 0.0001). Additionally, an OTU mapping to Cutibacterium acnes had a significantly higher relative abundance in PMA-treated (viable) samples. These results demonstrate that the total bacterial DNA content of human milk is not representative of the viable human milk microbiome. Our findings raise questions about the validity of conclusions drawn from previous studies in which viability testing was not used, and have broad implications for the design of future work in this field.

Detection of viable Lacticaseibacillus paracasei in fermented milk using propidium monoazide combined with quantitative loop-mediated isothermal amplification.

To quantify viable probiotic Lacticaseibacillus paracasei (L. paracasei) in fermented milk accurately and quickly, propidium monoazide combined with quantitative loop-mediated isothermal amplification (PMA-qLAMP) was applied. The optimal PMA treatment conditions for treating a L. paracasei suspension were determined using an orthogonal test to eliminate the DNA amplification of 108 CFU/mL of dead L. paracasei. Primers were designed based on the species-specific gyrB gene of L. paracasei. A phylogenetic tree based on the gyrB gene showed that L. paracasei clustered on the same branch with 91% support. Compared with the 16 strains commonly found in fermented milk, three strains of L. paracasei showed positive PMA-qLAMP results, and the melting temperature was approximately 82.4°C. There was a linear relationship (R2 = 0.9983) between the Ct values and the logarithm of the concentration of viable bacteria. The PMA-qLAMP detection limit for the L. paracasei artificially added to fermented milk was 7.3 × 102 CFU/mL. There was no significant difference between the logarithm values of the concentration of viable L. paracasei of 50 fermented milk samples within shelf life using the PMA-qLAMP and plate count methods (P > 0.01). PMA-qLAMP is specific and accurate for obtaining reliable results faster than when using plate counts.

Enumeration of Viable Non-Culturable Vibrio cholerae Using Droplet Digital PCR Combined With Propidium Monoazide Treatment.

Many bacterial species, including Vibrio cholerae (the pathogen that causes cholera), enter a physiologically viable but non-culturable (VBNC) state at low temperature or in conditions of low nutrition; this is a survival strategy to resist environmental stress. Identification, detection, and differentiation of VBNC cells and nonviable cells are essential for both microbiological study and disease surveillance/control. Enumeration of VBNC cells requires an accurate method. Traditional counting methods do not allow quantification of VBNC cells because they are not culturable. Morphology-based counting cannot distinguish between live and dead cells. A bacterial cell possesses one copy of the chromosome. Hence, counting single-copy genes on the chromosome is a suitable approach to count bacterial cells. In this study, we developed quantitative PCR-based methods, including real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), to enumerate VBNC V. cholerae cells by counting the numbers of single-copy genes in samples during VBNC-state development. Propidium monoazide (PMA) treatment was incorporated to distinguish dead cells from viable cells. Both PCR methods could be used to quantify the number of DNA copies/mL and determine the proportion of dead cells (when PMA was used). The methods produced comparable counts using three single-copy genes (VC1376, thyA, and recA). However, ddPCR showed greater accuracy and sensitivity than qPCR. ddPCR also allows direct counting without the need to establish a standard curve. Our study develops a PMA-ddPCR method as a new tool to quantify VBNC cells of V. cholerae. The method can be extended to other bacterial species.

The effects of removing dead bacteria by propidium monoazide on the profile of salivary microbiome.

BACKGROUND: Oral microbiome played an important role in maintaining healthy state and might exhibit certain changes under circumstances of diseases. However, current microbiological research using sequencing techniques did not regard dead bacteria as a separate part, causing findings based on subsequent analyses on dynamic equilibrium and functional pathways of microbes somewhat questionable. Since treatment by propidium monoazide (PMA) was able to remove dead bacteria effectively, it would be worth studying how the sequencing results after PMA treatment differed from those focusing on the whole microbiota. METHODS: Unstimulated whole saliva samples were obtained from 18 healthy people from 3 age groups (children, adults, and the elderly). After removal of dead bacteria by propidium monoazide (PMA), changes in the profile of salivary microbiome were detected using 16S rRNA sequencing technology, and differences among age groups were compared subsequently. RESULTS: Dead bacteria accounted for nearly a half of the whole bacteria flora in saliva, while freezing had little effect on the proportion of deaths. After treatment with PMA, the numbers of OTUs reduced by 4.4-14.2%, while the Shannon diversity indices decreased significantly (P < 0.01). Only 35.2% of positive and 6.1% of negative correlations were found to be shared by the whole microbiota and that with dead bacteria removed. Differences in significantly changed OTUs and functional pathways among different age groups were also observed between the group of PMA and the control. CONCLUSIONS: It was necessary to take the influence of living state of bacteria into account in analytic studies of salivary microbiome.

Microbiological testing of clinical samples before and after periodontal treatment. A comparative methodological study between real-time PCR and real-time-PCR associated to propidium monoazide.

OBJECTIVES: The aim of the present methodological study was to evaluate the discrepancies in the detection of a number of periodontally involved pathogenic bacteria obtained from clinical samples by two methods: the quantitative Polymerase Chain Reaction (qPCR) and the qPCR combined with pre-treatment by Propidium Monoazide (PMA). MATERIAL AND METHODS: Plaque and saliva samples were obtained from 30 subjects: 20 subjects with chronic or aggressive periodontitis in need of periodontal therapy with or without antibiotics and 10 subjects in Supportive Periodontal Treatment (SPT). The clinical samples taken before treatment (BL) and 1 month later (M1), were divided in two aliquots: one was immediately treated with PMA while the other was left untreated. All samples were further analyzed with qPCR after DNA extraction, for the detection of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Parvimonas micra (Pm), and Prevotella intermedia (Pi). RESULTS: Large inter-individual variations were observed in the concentration of the studied bacteria. At both instances (BL and M1) and for the three groups, significantly lower counts of bacteria were depicted when plaque and saliva samples were pre-treated with PMA as compared to those without treatment. Treatment resulted in significant decreases in the number of bacteria, mainly in the plaque samples. However, these changes were almost similar in the three groups independently of the method of detection used (PMA-qPCR vs. q-PCR). CONCLUSION: Removal of DNA from non-viable cells with PMA treatment is an easily applied step added to the classical qPCR that could give accurate information on the presence of viable bacterial load and evaluate the response to periodontal treatment.

Quantitative and specific detection of viable pathogens on a portable microfluidic chip system by combining improved propidium monoazide (PMAxx) and loop-mediated isothermal amplification (LAMP).

An accurate and specific detection of viable Candida albicans (C. albicans) in vaginal discharge is crucial for the diagnosis of vulvovaginal candidiasis (VVC) and assessment of antifungal effects. In this study, improved propidium monoazide (PMAxx) and loop-mediated isothermal amplification (LAMP) were used for the first time to distinguish between viable and dead C. albicans. A portable microfluidic chip system was developed to detect multiple viable pathogens in parallel. The consumption of samples and reagents in per reaction cell were only 0.94 µL, less than 1/25 of the conventional 25 µL Eppendorf tubular test method, both significantly reducing testing cost and greatly simplifying the detection of multiple viable pathogens. The concentration of PMAxx was optimized against C. albicans at 4.0 log CFU mL-1 to 5.0 log CFU mL-1, and 1 µM PMAxx was proven to be suitable for the detection of C. albicans in clinical samples. When testing mixtures containing different ratios of viable to dead C. albicans, PMAxx-LAMP could circumvent the signal arising from dead cells and, therefore, reflected the abundance of viable cells precisely. Furthermore, the suitability of this technique to evaluate the effects of antifungal agents, including clotrimazole, miconazole, and tioconazole, was assessed. Finally, the viability of Escherichia coli (E. coli) and C. albicans were detected on the portable microfluidic chip system. PMAxx-LAMP based portable microfluidic chip system was determined to be a feasible technique for assessing the viability of multiple pathogens in gynecology and might provide insights into new VVC treatment strategies.

Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR.

The ongoing COVID-19 pandemic has generated a global health crisis that needs well management of not only patients but also environments to reduce SARS-CoV-2 transmission. The gold standard RT-qPCR method is sensitive and rapid to detect SARS-CoV-2 nucleic acid, but does not answer if PCR-positive samples contain infectious virions. To circumvent this problem, we report an SDS-propidium monoazide (PMA) assisted RT-qPCR method that enables rapid discrimination of live and dead SARS-CoV-2 within 3 h. PMA, a photo-reactive dye, can react with viral RNA released or inside inactivated SARS-CoV-2 virions under assistance of 0.005% SDS, but not viral RNA inside live virions. Formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, RT-qPCR detection of heat-inactivated SARS-CoV-2 resulted in larger than 9 Ct value differences between PMA-treated and PMA-free groups, while less than 0.5 Ct differences were observed in the detection of infectious SARS-CoV-2 ranging from 20 to 5148 viral particles. Using a cutoff Ct difference of 8.6, this method could differentiate as low as 8 PFU live viruses in the mixtures of live and heat-inactivated virions. Further experiments showed that this method could successfully monitor the natural inactivation process of SARS-CoV-2 on plastic surfaces during storage with comparable results to the gold standard plaque assay. We believe that the culture-free method established here could be used for rapid and convenient determination of infectious SARS-CoV-2 virions in PCR-positive samples, which will facilitate better control of SARS-CoV-2 transmission.

Changes in physico-chemical characteristics and viable bacterial communities during fermentation of alfalfa silages inoculated with Lactobacillus plantarum.

This study investigated the effect of inoculating Lactobacillus (L.) plantarum PS-8 in fermentation of alfalfa silages. We monitored the fermentation characteristics and bacterial population dynamics during the ensiling process. PacBio single molecule real time sequencing was combined with propidium monoazide (PMA) treatment to monitor the viable microbiota dynamics. We found that inoculating L. plantarum PS-8 may improve the silage quality by accelerating acidification, reducing the amounts of clostridia, coliform bacteria, molds and yeasts, elevating the protein and organic acid contents (except butyrate), and enhancing lactic acid bacteria (LAB) while suppressing harmful microorganisms. Some significant differential abundant taxa were found between the PMA-treated and non-treated microbiota. For example, the relative abundances of L. brevis, L. plantarum, and Pediococcus pentosaceus were significantly higher in the PMA-treated group than the non-PMA-treated group, suggesting obvious differences between the viable and non-viable microbiota. It would thus be necessary to distinguish between the viable and non-viable microbial communities to further understand their physiological contribution in silage fermentation. By tracking the dynamics of viable microbiota in relation with changes in the physico-chemical parameters, our study provided novel insights into the beneficial effects of inoculating L. plantarum PS-8 in silage fermentation and the physiological function of the viable bacterial communities.

Rapid determination of viable but non-culturable Campylobacter jejuni in food products by loop-mediated isothermal amplification coupling propidium monoazide treatment.

Campylobacter is the leading cause of foodborne human diarrhea worldwide. This microbe in the viable but non-culturable (VBNC) state can evade detection by routinely used culture-based methods and remain viable for extended periods of time. Bacteria in this dormancy state can resume their metabolic activity and virulence by resuscitation under favorable conditions, and subsequently cause infections. In this study, an assay combining loop-mediated isothermal amplification (LAMP) and propidium monoazide (PMA) treatment was developed for the detection and quantification of VBNC C. jejuni in agri-foods. PMA-qLAMP targeting the hipO gene demonstrated 100% high specificity to C. jejuni. A linear detection of C. jejuni was achieved between 8.77 × 102 and 8.77 × 07 CFU/mL with a coefficient of determination (R2) of 0.9956, indicating a good quantitative capacity. C. jejuni was effectively induced into the VBNC state by osmotic stress (i.e., 7% NaCl, w/v) over 48 h. VBNC C. jejuni cells were spiked into three representative food products and determined by PMA-qLAMP coupled with plating assay. The detection limits of PMA-qLAMP were 1.58 × 102 CFU/mL in milk, 3.78 × 102 CFU/g in chicken breast meat, and 4.33 × 102 CFU/g in romaine lettuce. PMA-qLAMP demonstrated rapid (25-40 min), specific (100% inclusivity and 100% exclusivity) and sensitive (~102 CFU/mL) determination of VBNC C. jejuni. This method can be applied in the agri-food industry to decrease the risks related to the consumption of contaminated agri-foods with pathogenic bacteria in the VBNC state and reduce the burden of C. jejuni infections to public health.

Quantification of viable protozoan parasites on leafy greens using molecular methods.

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.