Propidium Monoazide Treatment Research Articles

Shifts and importance of viable bacteria in treatment of DSS-induced ulcerative colitis mice with FMT.

Ulcerative colitis (UC) has become a global public health concern, and is in urgent need of novel therapies. Fecal microbiota transplantation (FMT) targeting gut microbiota has recently been applied to the treatment of UC. Despite its recent successes, it is still largely unknown how FMT functionally modulates the gut microbiota and improves the disease. We prospectively collected fecal samples from the 40 mice (30 mice for dextran sulfate sodium (DSS)-induced, 10 for controls), followed by Propidium monoazide treatment for 16S rRNA gene sequencing. These 30 mice were divided equally into 3 groups, which were transplanted with original donor microbiota (DO), inactivated donor microbiota (DI) and saline, respectively. Subsequently, we used 16S rRNA gene sequencing to analyze the viable gut bacteria of ulcerative colitis (UC) mice and histological analysis to evaluate the effects of fecal microbiota transplantation (FMT) with viable microbiota. We demonstrated that the community structure of viable bacteria was significantly different from fecal bacteria based on total DNA. Furthermore, the intestinal viable microbiota and colonic mucosal structure of mice were significantly changed by DSS induction. The histological analysis showed that only the mice treated with original donor microbiota group (HF) achieved a significant improvement. Compared with inactivated donor microbiota group (IF) and saline (NF), Lactobacillus and Halomonas were significantly enriched in the HF group. We inferred that only live bacteria from human donor reversed the histopathology and symptoms of UC in mice and altered the gut microbiota. The activity of gut microbiota in donor samples should be considered in FMT and that detailed analysis of viable microbiota is essential to understand the mechanisms by which FMT produces therapeutic effects in the future.

Evaluation of Bacterial Composition and Viability of Equine Feces after Processing for Transplantation

Fecal microbiota transplantation (FMT) has been used empirically for decades in equine medicine to treat intestinal dysbiosis but evidence-based information is scarce. This in vitro study aimed at assessing the effect of a commonly used pre-FMT processing method on the bacterial composition and viability of the fecal filtrate. Three samples of fresh equine manure (T0) were processed identically: the initial manure was mixed with 1 L of lukewarm water and chopped using an immersion blender to obtain a mixture (T1), which was left uncovered during 30 min (T2) and percolated through a sieve to obtain a fecal filtrate (T3). Samples were taken throughout the procedure (Tn) and immediately stored at 4 °C until processing. The 16S rDNA amplicon profiling associated with propidium monoazide treatment was performed on each sample to select live bacteria. Analyses of α and β diversity and main bacterial populations and quantitative (qPCR) analysis were performed and statistically compared (significance p < 0.05) between time points (T0-T3). No significant differences in ecological indices or mean estimated total living bacteria were found in the final fecal filtrate (T3) in regard to the original manure (T0); however, relative abundances of some minor genera (Fibrobacter, WCHB1-41_ge and Akkermansia) were significantly different in the final filtrate. In conclusion, the results support the viability of the major bacterial populations in equine feces when using the described pre-FMT protocol.

An improved workflow for accurate and robust healthcare environmental surveillance using metagenomics

BackgroundEffective surveillance of microbial communities in the healthcare environment is increasingly important in infection prevention. Metagenomics-based techniques are promising due to their untargeted nature but are currently challenged by several limitations: (1) they are not powerful enough to extract valid signals out of the background noise for low-biomass samples, (2) they do not distinguish between viable and nonviable organisms, and (3) they do not reveal the microbial load quantitatively. An additional practical challenge towards a robust pipeline is the inability to efficiently allocate sequencing resources a priori. Assessment of sequencing depth is generally practiced post hoc, if at all, for most microbiome studies, regardless of the sample type. This practice is inefficient at best, and at worst, poor sequencing depth jeopardizes the interpretation of study results. To address these challenges, we present a workflow for metagenomics-based environmental surveillance that is appropriate for low-biomass samples, distinguishes viability, is quantitative, and estimates sequencing resources.ResultsThe workflow was developed using a representative microbiome sample, which was created by aggregating 120 surface swabs collected from a medical intensive care unit. Upon evaluating and optimizing techniques as well as developing new modules, we recommend best practices and introduce a well-structured workflow. We recommend adopting liquid-liquid extraction to improve DNA yield and only incorporating whole-cell filtration when the nonbacterial proportion is large. We suggest including propidium monoazide treatment coupled with internal standards and absolute abundance profiling for viability assessment and involving cultivation when demanding comprehensive profiling. We further recommend integrating internal standards for quantification and additionally qPCR when we expect poor taxonomic classification. We also introduce a machine learning-based model to predict required sequencing effort from accessible sample features. The model helps make full use of sequencing resources and achieve desired outcomes.DVg4DtPBM9AbUXUoj_2n2mVideo ConclusionsThis workflow will contribute to more accurate and robust environmental surveillance and infection prevention. Lessons gained from this study will also benefit the continuing development of methods in relevant fields.

Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study.

Although high-throughput DNA sequencing-based methods have been of great value for determining the composition of microbial communities in various environments, there is the potential for inaccuracies arising from the sequencing of DNA from dead microorganisms. In this pilot study, we compared different sequencing-based methods to assess their relative accuracy with respect to distinguishing between viable and non-viable cells, using a live and heat-inactivated model community spiked into bovine milk. The methods used were shotgun metagenomics with and without propidium monoazide (PMA) treatment, RNA-based 16S rRNA sequencing and metatranscriptomics. The results showed that methods were generally accurate, though significant differences were found depending on the library types and sequencing technologies. Different molecular targets were the basis for variations in the results generated using different library types, while differences in the derived composition data from Oxford Nanopore Technologies-and Illumina-based sequencing likely reflect a combination of different sequencing depths, error rates and bioinformatics pipelines. Although PMA was successfully applied in this study, further optimisation is required before it can be applied in a more universal context for complex microbiomes. Overall, these methods show promise and represent another important step towards the ultimate establishment of approaches that can be applied to accurately identify live microorganisms in milk and other food niches.

Quantitative detection of Vibrio parahaemolyticus in aquatic products by duplex droplet digital PCR combined with propidium monoazide

Based on the combination of droplet digital PCR (ddPCR) and propidium monoazide (PMA), a new method called PMA-duplex ddPCR was developed for the quantitative determination of viable Vibrio parahaemolyticus in aquatic products. Specific primers and probes targeting a new gene, vp0488, coding for putative DNA polymerase III beta chain, which was self-screened by this laboratory, and a traditional target gene, thermolabile hemolysin (tlh), were designed and verified. The duplex ddPCR system and conditions and PMA treatment conditions were optimized. The limit of detection (LOD) for PMA-duplex ddPCR was evaluated and applied to detect V. parahaemolyticus in artificially contaminated samples and actual samples. The results indicate that the optimal duplex ddPCR annealing temperature was 60 °C, the optimal number of amplification cycles was 40, and the optimal ratio of primer to probe was 1:0.5. The optimal PMA treatment conditions were as follows: incubation time 5 min, concentration 30 μmol/L, and exposure time 15 min. The LOD of PMA-duplex ddPCR (7.32 × 101 CFU/mL) was lower than that of duplex quantitative PCR (qPCR) (7.32 × 102 CFU/mL). The lowest detectable V. parahaemolyticus in oysters was 8.15 × 101 CFU/g without any pre-enrichment. The method established in this study has the advantages of good specificity, high sensitivity and accuracy, which provides an effective method for the detection of viable Vibrio parahaemolyticus in aquatic foods.

Dynamics of Starter and Non-Starter Lactic Acid Bacteria Populations in Long-Ripened Cheddar Cheese Using Propidium Monoazide (PMA) Treatment.

The microbial community of industrially produced Canadian Cheddar cheese was examined from curd to ripened cheese at 30–32 months using a combination of viable plate counts of SLAB (GM17) and NSLAB (MRSv), qPCR and 16S rRNA gene amplicon sequencing. Cell treatment with propidium monoazide excluded DNA of permeable cells from amplification. The proportion of permeable cells of both Lactococcus spp. and Lacticaseibacillus spp. was highest at 3–6 months. While most remaining Lacticaseibacillus spp. cells were intact during later ripening stages, a consistent population of permeable Lactococcus spp. cells was maintained over the 32-month period. While Lactococcus sequence variants were significant biomarkers for viable cheese curd communities at 0–1 m, Lacticaseibacillus was identified as a distinctive biomarker for cheeses from 7 to 20 months. From 24 to 32 months, Lacticaseibacillus was replaced in significance by four genera (Pediococcus and Latilactobacillus at 24 m and at 30–32 m, Secundilactobacillus and Paucilactobacillus). These results underscore the importance of monitoring potential defects in cheeses aged over 24 months, which could be diagnosed early through microbial DNA profiling to minimize potential waste of product. Future perspectives include correlating volatile flavor compounds with microbial community composition as well as the investigation of intra-species diversity.

Cryopreservation of stool samples altered the microbial viability quantitively and compositionally.

Stool is the most commonly used sample for gut microbiota analysis in humans and animals. Cryopreservation of stool at -80°C is a feasible and simple method in clinics and researches, especially in large-scale cohort studies. However, the viability of bacteria in stool after freezing has yet well-demonstrated quantitatively and compositionally. This study determined the viable microbiota of samples under cryopreservation at -80°C, relative to fresh samples and that stored at ambient. Stool samples were collected from three healthy adults. Propidium monoazide treatment combined with quantitative PCR and 16S rRNA gene sequencing was performed to target viable microbiota. After freezing, the number of viable bacteria decreased, though inter-individual difference existed. Notably, the alpha diversity of viable microbiota after freezing did not change significantly, while its composition changed. Freezing significantly reduced the viable bacteria in Gram-negative genera of Bacteroidetes and Firmicutes, and proportionally increased Gram-positive bacteria in genera of Actinobacteria and Firmicutes, including Bifidobacterium, Collinsella and Blautia, implying that the cell envelope structure associated with the bacterial sensitivity to freezing. On the contrary, the room temperature storage not only decreased the number of viable bacteria, but also decreased the microbial alpha diversity, and remarkably enriched facultative anaerobes of Escherichia-Shigella, Enterococcus and Lactococcus, some of which are opportunistic pathogens. Our findings suggested that changes in viable microbiota in stool samples caused by cryopreservation should be paid enough attention for downstream utilization.

An Assay Combining Droplet Digital PCR With Propidium Monoazide Treatment for the Accurate Detection of Live Cells of Vibrio vulnificus in Plasma Samples.

Vibrio vulnificus (V. vulnificus) is one of the most common pathogenic Vibrio species to humans; therefore, the establishment of timely and credible detection methods has become an urgent requirement for V. vulnificus illness surveillance. In this study, an assay combining droplet digital PCR (ddPCR) with propidium monoazide (PMA) treatment was developed for detecting V. vulnificus. The primers/probes targeting the V. vulnificus hemolysin A (vvhA) gene, amplification procedures, and PMA processing conditions involved in the assay were optimized. Then, we analyzed the specificity, sensitivity, and ability to detect live cell DNA while testing the performance of PMA-ddPCR in clinical samples. The optimal concentrations of primers and probes were 1.0 and 0.3 μM, respectively. The annealing temperature achieving the highest accuracy in ddPCR assay was 60°C. With an initial V. vulnificus cell concentration of 108 CFU/mL (colony-forming units per milliliter), the optimal strategy to distinguish live cells from dead cells was to treat samples with 100 μM PMA for 15 min in the dark and expose them to LED light with an output wavelength of 465 nm for 10 min. The specificity of the PMA-ddPCR assay was tested on 27 strains, including seven V. vulnificus strains and 20 other bacterial strains. Only the seven V. vulnificus strains were observed with positive signals in specificity analysis. Comparative experiments on the detection ability of PMA-ddPCR and PMA-qPCR in pure cultures and plasma samples were performed. The limit of detection (LOD) and the limit of quantitation (LOQ) in pure culture solutions of V. vulnificus were 29.33 and 53.64 CFU/mL in PMA-ddPCR, respectively. For artificially clinical sample tests in PMA-ddPCR, V. vulnificus could be detected at concentrations as low as 65.20 CFU/mL. The sensitivity of the PMA-ddPCR assay was 15- to 40-fold more sensitive than the PMA-qPCR in this study. The PMA-ddPCR assay we developed provides a new insight to accurately detect live cells of V. vulnificus in clinical samples, which is of great significance to enhance public health safety and security capability and improve the emergency response level for V. vulnificus infection.

Strategy to Evaluate Changes in Bacterial Community Profiles and Bacterial Pathogen Load Reduction After Sewage Disinfection.

Sewage effluent discharge is a major source of pathogenic contamination to the environment. The disinfection process is critical for the elimination of pathogens in sewage. In this study, we examined the impact of chlorine disinfection on the total, viable, and culturable populations of indicator bacteria, pathogens, and bacterial communities in two contrasting types of effluents (primarily treated saline and secondarily treated freshwater). Effluents collected bimonthly over 1 year were examined using cultivation, quantitative PCR (qPCR), and 16S rRNA gene amplicon sequencing coupled with or without propidium monoazide (PMA) treatment. The results showed that each type of effluent was characterized by a specific set of representative genera before disinfection. Salinity appeared to be the major abiotic factor associated with the differences in bacterial community compositions. The pathogen analysis pipeline revealed over 20 viable clinically important pathogenic species in the effluents. Although the bacterial communities differed markedly between the two types of effluents before disinfection, the species of pathogens persisting after disinfection were similar, many of them were members of Enterobacter and Vibrio. The relative abundances of all pathogens identified in the amplicon sequences were multiplied by the 16S rRNA gene copy numbers of total bacteria detected by PMA-qPCR to estimate their concentrations. Pathogens remained viable after disinfection reached 8 log10 16S rRNA copies ml−1 effluent. Meanwhile, around 80 % of the populations of three indicator bacteria including Escherichia coli, Enterococcus, and Bacteroidales were viable after disinfection, but over 99 % of the viable E. coli and Enterococcus were in the non-culturable state. We estimated the total pathogen load by adding the concentrations of all viable pathogens and examined their correlations with indicator bacteria of different types, physiological states, and effluents. The results showed that the PMA-qPCR measurement of E. coli is a reliable proxy of bacterial pathogen loads in both types of effluents. The utility of viable indicator bacteria as a biological index to assess the overall bacteriological hazards in effluents is discussed.

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.

Simultaneous detection of viable Salmonella spp., Escherichia coli, and Staphylococcus aureus in bird's nest, donkey-hide gelatin, and wolfberry using PMA with multiplex real-time quantitative PCR.

Salmonella spp., Escherichia coli, and Staphylococcus aureus are common microbial contaminants within the homology of medicine and food that can cause serious food poisoning. This study describes a highly efficient, sensitive, specific, and simple multiplex real‐time quantitative PCR (mRT‐qPCR) method for the simultaneous detection of viable Salmonella spp., E. coli, and S. aureus. Primers and probes were designed for the amplification of the target genes invA, uidA, and nuc. Dead bacterial genetic material was excluded by propidium monoazide (PMA) treatment, facilitating the detection of only viable bacteria. This method was capable of detecting Salmonella spp., E. coli, and S. aureus at 102, 102, and 101 CFU/ml, respectively, in pure culture. PMA combined with mRT‐qPCR can reliably distinguish between dead and viable bacteria with recovery rates from 95.7% to 105.6%. This PMA‐mRT‐qPCR technique is a highly sensitive and specific method for the simultaneous detection of three pathogens within the homology of medicine and food.

Rapid molecular diagnosis of live Mycobacterium tuberculosis on an integrated microfluidic system

Tuberculosis (TB) is a serious, potentially fatal disease that has been a global public health issue for centuries. The growth of the causal bacterium Mycobacterium tuberculosis (M. tuberculosis) is slow (at least 6–8 weeks) when compared to other infectious bacteria, thus posing a challenge for early diagnostics. Furthermore, TB therapy has been reported to be effective when antibiotics are administered during the early stage. Therefore, accurate and rapid diagnosis of M. tuberculosis plays an important role in TB quarantine and treatment. We therefore devised an integrated microfluidic system to automatically detect live M. tuberculosis and distinguish dead bacteria. A heparin-binding hemagglutinin antibody was used to capture bacteria within 10 min. The photo-reactive dye propidium monoazide was then used to bind double-stranded DNA from dead bacteria with high affinity (20 min) such that genes of dead bacteria were not amplified by the subsequent PCR. Additionally, after 10-min of genomic DNA isolation, the final PCR step (50 min) was carried out to detect live M. tuberculosis. It is the first time that the entire bacterial detection process (including bacterial capture, propidium monoazide treatment, lysis and PCR quantification) for live M. tuberculosis could be automated within 90 min on a single chip with a limit of detection as low as 100 colony-forming units. This integrated microfluidic system is promising for point-of-care diagnostics given its automation, high speed, low cost, low sample/reagent consumption, and reduced risk of infection for front-line medical staff.

Two-Round Treatment With Propidium Monoazide Completely Inhibits the Detection of Dead Campylobacter spp. Cells by Quantitative PCR.

Campylobacter spp. are known as important foodborne gastroenteric pathogens worldwide. Campylobacter spp. can exist in a viable but non-culturable (VBNC) state under unsuitable environmental conditions, which is undetectable by conventional culture methods. Quantitative polymerase chain reaction (qPCR) can be used to detect VBNC Campylobacter spp.; however, both viable and dead bacteria are detected during qPCR and are indistinguishable. Propidium monoazide (PMA), which can only enter dead bacterial cells through a damaged cell wall/cell membrane, binds to DNA and inhibits qPCR. PMA treatment has been performed along with qPCR (PMA-qPCR) to detect viable bacteria. However, the efficacy of detection inhibition differed among studies, and PMA can potentially enter living cells after changes in cell membrane permeability. In this study, we optimized the PMA treatment method by conducting it before qPCR. Two-round PMA treatment completely inhibited the qPCR signals from dead cells, whereas single-round PMA treatment failed to facilitate this. An optimized PMA-qPCR method was developed using commercial chicken meat, and VBNC Campylobacter spp., which are undetectable using conventional culture-based methods, were successfully detected. In conclusion, this study presents a novel, efficient PMA treatment method for the detection of viable Campylobacter spp., including VBNC Campylobacter spp., in chicken meat. We believe that this method will aid the reliable risk assessment of commercial chicken meat.

Changes in physiological states of Salmonella Typhimuriummeasured 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 Typhimuriuminduced 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. Typhimuriumafter the pasteurization and UV treatments, while viability PCR with propidium monoazide (PMA) and DyeTox13-qPCR indicated that the membrane integrity of S. Typhimuriumremained 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.

BackgroundPolymerase 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.MethodsVarying 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.ResultsPMA’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.ConclusionWe 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.

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.

Assessment of Comamonas testosteroni strain PT9 as a rapid phthalic acid degrader for industrial wastewaters.

In this study, characterization of industry-borne Comamonas testosteroni strain PT9 isolate was performed by determining degradation ability on phthalic acid (PA). High-performance liquid chromatography analyses showed that strain PT9 completely degraded 102.94 mg/L of PA within 6 h. Viability polymerase chain reaction (vPCR) was performed with propidium monoazide treatment. vPCR showed that the PA has positively stimulated the cell growth during degradation. To consider the fate of PA, the proposed catalytic genes (ophA2, iphA2, tphA2, tphA3, pmdA, and pmdB) for the degradation pathways of PA isomers for C. testosteroni were screened in strain PT9. All genes except iphA2 were detected in strain PT9, and expression levels of related genes were analyzed by Real-Time PCR (qPCR).

Challenging the “gold standard” of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the “gold standard” in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log10Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination.Campylobacter served as a model organism, challenging CFU as “gold standard” and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens.

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

BackgroundOral 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.MethodsUnstimulated 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.ResultsDead 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.ConclusionsIt was necessary to take the influence of living state of bacteria into account in analytic studies of salivary microbiome.

Viability-Resolved Metagenomics Reveals Antagonistic Colonization Dynamics of Staphylococcus epidermidis Strains on Preterm Infant Skin.

ABSTRACTPreterm infants are at increased risk of infections caused by coagulase-negative staphylococci (CoNS) that colonize skin. Technical barriers in sequencing low-microbial-biomass skin swabs from preterm infants hinder attempts to gain a strain-level understanding of CoNS colonization dynamics within their developing skin microbiome. Here, the microbiome of five skin sites and available stool was studied from four preterm infants hospitalized over their first 2 months of life. We used propidium monoazide treatment of samples to enrich for the viable microbiome and metagenomic shotgun sequencing to resolve species and strains. The microbiome of different skin sites overlapped with each other, was dominated by the CoNS species Staphylococcus epidermidis and Staphylococcus capitis, and was distinct from stool. Species diversity on skin increased over time despite antibiotic exposure. Evidence of antagonism between the most common S. epidermidis strains, ST2 and ST59, included negative relationships for species correlation networks and in situ replication rates and that ST2 colonized skin earlier but was often replaced by ST59 over time. Experiments done with reference isolates showed that ST2 produced more biofilm than ST59 on plastic surfaces, which was reduced in mixed culture. We also discovered that a rare S. epidermidis strain, ST5, grew rapidly in stool in association with Stenotrophomonas maltophilia from a suspected episode of infection. Viability treatment of samples and moderate throughput shotgun sequencing provides strain-level information about CoNS colonization dynamics of preterm infant skin that ultimately might be exploited to prevent infections.IMPORTANCE The skin is a habitat for microbes that commonly infect preterm infants, but the use of sequencing for fine-scale study of the microbial communities of skin that develop in these infants has been limited by technical barriers. We treated skin swabs of preterm infants with a photoreactive dye that eliminates DNA from nonviable microbes and then sequenced the remaining DNA. We found that two strains of the most common species, Staphylococcus epidermidis, showed an antagonistic relationship on skin by cooccurring with different species, replicating fastest in different samples, and dominating skin sites at different times. Representatives of these strains also differed in their ability to stick to plastic surfaces—an important pathogenicity trait of this species. Our study shows the feasibility of gaining detailed information about strain colonization dynamics from this difficult-to-sequence body site of preterm infants, which might be used to guide novel approaches to prevent infections.