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.

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.

Recovery of Mycobacteria from Heavily Contaminated Environmental Matrices

For epidemiology studies, a decontamination method using a solution containing 4.0% NaOH and 0.5% tetradecyltrimethylammonium bromide (TDAB) represents a relatively simple and universal procedure for processing heavily microbially contaminated matrices together with increase of mycobacteria yield and elimination of gross contamination. A contamination rate only averaging 7.3% (2.4% in Cluster S; 6.9%% in Cluster R and 12.6%% in Cluster E) was found in 787 examined environmental samples. Mycobacteria were cultured from 28.5% of 274 soil and water sediments samples (Cluster S), 60.2% of 251 samples of raw and processed peat and other horticultural substrates (Cluster R), and 29.4% of 262 faecal samples along with other samples of animal origin (Cluster E). A total of 38 species of slow and rapidly growing mycobacteria were isolated. M. avium ssp. hominissuis, M. fortuitum, and M. malmoense were the species most often isolated. The parameters for the quantitative detection of mycobacteria by PCR can be significantly refined by treating the sample suspension before DNA isolation with PMA (propidium monoazide) solution. This effectively eliminates DNA residue from both dead mycobacterial cells and potentially interfering DNA segments present from other microbial flora. In terms of human exposure risk assessment, the potential exposure to live non-tuberculous mycobacteria can be more accurately determined.

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: PMAs 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 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.

Free DNA and Metagenomics Analyses: Evaluation of Free DNA Inactivation Protocols for Shotgun Metagenomics Analysis of Human Biological Matrices

Culture-independent approaches now represent the gold standard for the investigation of both environmental and host-associated complex microbial communities. Nevertheless, despite the great advantages offered by these novel methodologies based on the use of next-generation DNA sequencing approaches, a number of bias sources have been identified. Among the latter, free DNA contained in biological matrices is one of the main sources of inaccuracy in reconstructing the resident microbial population of viable cells. For this reason, the photoreactive DNA-binding dye propidium monoazide (PMAxx™) has been developed by improving standard PMA. This compound binds and inactivates free DNA, thus preventing its amplification and sequencing. While the performances of PMA have been previously investigated, the efficiency with PMAxx™ has been tested mainly for amplicon-based profiling approaches on a limited number of biological matrices. In this study, we validated the performance of PMAxx™ for shotgun metagenomics approaches employing various human-associated matrices. Notably, results revealed that the effectiveness of PMAxx™ in inactivating free DNA of prokaryotes and eukaryotes tends to vary significantly based on the biological matrices analyzed.

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.

Fate of Functional Bacterial and Eukaryotic Community Regulated by Earthworms during Vermicomposting of Dewatered Sludge, Studies Based on the 16S rDNA and 18S rDNA Sequencing of Active Cells

DNA sequencing of active cells involved in vermicomposting can clarify the roles of earthworms in regulating functional microorganisms. This study aimed to investigate the effect of earthworms on functional microbial communities in sludge by comparing biodegradation treatments with and without earthworms. PCR and high throughput sequencing based on pretreatment of propidium monoazide (PMA) were used to detect the changes in active bacterial 16S rDNA and eukaryotic 18S rDNA during vermicomposting. The results showed that the nitrate in sludge vermicomposting and control were significantly different from day 10, with a more stable product at day 30 of vermicomposting. Compared with the control, the Shannon indexes of active bacteria and eukaryotes decreased by 1.9% and 31.1%, respectively, in sludge vermicompost. Moreover, Proteobacteria (36.2%), Actinobacteria (25.6%), and eukaryotic Cryptomycota (80.3%) were activated in the sludge vermicompost. In contrast, the control had Proteobacteria (44.8%), Bacteroidetes (14.2%), Cryptomycota (50.00%), and Arthropoda (36.59%). Network analysis showed that environmental factors had different correlations between active bacterial and eukaryotic community structures. This study suggests that earthworms can decrease the diversity of bacterial and eukaryotic communities, forming a specific-functional microbial community and thus accelerating organic matter decomposition during vermicomposting of dewatered sludge.

Impact of pH and removed filtrate on E. coli regrowth and microbial community during storage of electro-dewatered biosolids

ABSTRACTResidual biosolids can be land applied if they meet microbiological requirements at the time of application. Electro-dewatering technology is shown to reduce biosolids bacterial counts to detection limits with little potential for bacterial regrowth during incubations. Here, we investigated the impacts on Escherichia coli regrowth and microbial communities of biosolids pH, removed nutrients via the filtrate, and produced inhibitory compounds in electro-dewatered biosolids. Findings suggest pH as the primary mechanism impacting E. coli regrowth in electro-dewatered biosolids. Propidium monoazide treatments were effective to remove DNA from dead cells based on the removal of obligate anaerobes observed after anaerobic incubation. Analyses of high throughput sequenced data showed lower alpha-diversities associated with electro-dewatering treatment and incubation time. Moreover, biosolids pH and incubation period were the main factors contributing to the variations in microbial community compositions after incubation. Results highlight the role of biosolids pH on regrowing culturable bacteria and overall microbial communities.

Development of a Direct and Rapid Detection Method for Viable but Non-culturable State of Pediococcus acidilactici

Pediococcus acidilactici may significantly reduce the pH-value, and thus has different influence, including serving as a probiotic in human microbiota but a spoilage in human food as it could change the flavor. Pediococcus acidilactici is also capable of entering into the viable but non-culturable (VBNC) state causing false negative results of standard culture-based detection method. Thus, development of detection method for VBNC state P. acidilactici is of great significance. In this study, propidium monoazide (PMA) combined with cross priming amplification (CPA) was developed to detect the VBNC cells of P. acidilactici and applied on the detection in different systems. With detection limit of 104 cells/ml, high sensitivity, and 100% specificity, PMA-CPA can successfully detect VBNC cells of P. acidilactici and be applied in with high robustness.