Comparative analysis of the sensitivity of metagenomic sequencing and PCR to detect a biowarfare simulant (Bacillus atrophaeus) in soil samples

Plaire, Delphine; Puaud, Simon; Marsolier‐Kergoat, Marie‐Claude; Elalouf, Jean‐Marc

doi:10.1371/journal.pone.0177112

Cited by 16 publications

(13 citation statements)

References 47 publications

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“…Therefore, a negative culture result in screening for S. aureus or MRSA at a specific point in time does not completely eliminate the risk of infections with them. The low detection rate in nuc-PCR (36.9%) suggests that a conventional PCR is insensitive compared to real-time PCR and NGS methods, which exhibit about 100 to 1,000 times higher sensitivity than that of conventional PCR (54,55).…”

Section: Discussionmentioning

confidence: 99%

Development of a New Application for Comprehensive Viability Analysis Based on Microbiome Analysis by Next-Generation Sequencing: Insights into Staphylococcal Carriage in Human Nasal Cavities

Sasaki

Kuwahara‐Arai

et al. 2018

Appl Environ Microbiol

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The nasal carriage rate of in human is 25--30% and sporadically causes severe infections. However, mechanisms underlying staphylococcal carriage remain largely unknown. In the present study, we constructed an -based microbiome method for staphylococcal species discrimination. Based on a microbiome scheme targeting viable cell DNA using PMA dye (PMA microbiome), we also developed a new method to allow the comprehensive viability analysis of any bacterial taxon. To clarify the ecological distribution of staphylococci in the nasal microbiota, we applied these methods in 46 nasal specimens from healthy adults. PMA microbiome results showed that and were the most predominant viable taxa (average relative abundance: 0.435262 and 0.375195, respectively), and exhibited the highest viability in the nasal microbiota. detection rates from nasal specimens by-based conventional and PMA microbiome were 84.8% (39 of 46) and 69.5% (32 of 46), respectively, which substantially exceeded the values by a culture method using identical specimens (36.9%). Our results suggests that especially adapted most successfully to human nasal cavity. High detection of DNA by microbiome methods suggests that almost all healthy adults are consistently exposed to in everyday life. Furthermore, the large difference in detection rates between culture and microbiome methods suggests that cells frequently exist under the viable but non-culturable state in nasal cavities. Our method and findings will contribute to a better understanding of the mechanisms underlying carriage of indigenous bacteria. Metagenomic analyses, such as 16S rRNA microbiome, have provided new insight in various research fields. However, conventional 16S rRNA microbiome dose not permit taxonomic analysis of only viable bacteria, and has poor resolving power below the genus level. Our new schemes allowed for viabile cell specific analysis and species discrimination, nasal microbiome data using these methods provided some interesting findings regarding staphylococcal nasal carriage. According to our comprehensive viability analysis, high viability of genus, especialy in human nasal carriage suggests that this taxon has adapted most successfully to human nasal tissue. And also, higher detection of DNA by microbiome methods (84.8%) than that by a culture method (36.9%) suggests that almost all healthy adults are consistently exposed to in the medium- and long-term. Our findings will contribute to a better understanding of the mechanisms underlying carriage of indigenous bacteria.

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Section: Discussionmentioning

confidence: 99%

Development of a New Application for Comprehensive Viability Analysis Based on Microbiome Analysis by Next-Generation Sequencing: Insights into Staphylococcal Carriage in Human Nasal Cavities

Sasaki

Kuwahara‐Arai

et al. 2018

Appl Environ Microbiol

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“…The number of child stools detected positive for the Campylobacter genus was significantly higher using the MeTRS approach (88% [80-93.6%]) compared to the genus-specific PCR approach (50% [40-61%]; 16S RNA primer). Several studies showed that shotgun metagenomics and MeTRS had at least equal sensitivity to real-time PCR based approach for different pathogens (29,(35)(36)(37). Further, discrepancies between the two detection methods was observed, especially with the speciesspecific PCR approach (ceuE primer for C. coli and mapA primer for C. jejuni).…”

Section: Discussionmentioning

confidence: 99%

Co-occurrence of Campylobacter Species in Children From Eastern Ethiopia, and Their Association With Environmental Enteric Dysfunction, Diarrhea, and Host Microbiome

Terefe

Deblais

Ghanem

et al. 2020

Front. Public Health

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High Campylobacter prevalence during early childhood has been associated with stunting and environmental enteric dysfunction (EED), especially in low resource settings. This study assessed the prevalence, diversity, abundance, and co-occurrence of Campylobacter spp. in stools from children in a rural area of eastern Ethiopia and their association with microbiome, diarrhea, and EED in children. Stool samples (n = 100) were collected from randomly selected children (age range: 360-498 days) in five kebeles in Haramaya District, Ethiopia. Diarrhea, compromised gut permeability, and gut inflammation were observed in 48, 45, and 57% of children, respectively. Campylobacter prevalence and species diversity were assessed using PCR and meta-total RNA sequencing (MeTRS). The prevalence of Campylobacter spp. in the children's stools was 50% (41-60%) by PCR and 88% (80-93.6%) by MeTRS (P < 0.01). Further, seven Campylobacter species (Campylobacter jejuni, Campylobacter upsaliensis, Campylobacter hyointestinalis, Campylobacter coli, Campylobacter sp. RM6137, uncultured Campylobacter sp., and Campylobacter sp. RM12175) were detected by MeTRS in at least 40% of children stools in high abundance (>1.76-log read per million per positive stool sample). Four clusters of Campylobacter species (5-12 species per cluster) co-occurred in the stool samples, suggesting that Campylobacter colonization of children may have occurred through multiple reservoirs or from a reservoir in which several Campylobacter species may co-inhabit. No associations between Campylobacter spp., EED, and diarrhea were detected in this cross-sectional study; however, characteristic microbiome profiles were identified based on the prevalence of Campylobacter spp., EED severity, and diarrhea. Forty-seven bacterial species were correlated with Campylobacter, and 13 of them also correlated with gut permeability, gut inflammation and/or EED severity. Forty-nine species not correlated with Campylobacter Terefe et al. Campylobacter and Microbiome in Stools were correlated with gut permeability, gut inflammation, EED severity and/or diarrhea. This study demonstrated that (1) in addition to C. jejuni and C. coli, multiple nonthermophilic Campylobacter spp. (i.e., Campylobacter hyointestinalis, Campylobacter fetus, and Campylobacter concisus) were frequently detected in the children's stools and (2) the Campylobacter, gut permeability, gut inflammation, EED severity, and diarrhea were associated with characteristic microbiome composition. Additional spatial and longitudinal studies are needed to identify environmental reservoirs and sources of infection of children with disparate Campylobacter species and to better define their associations with EED in low-income countries.

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“…In the case that multiple ASVs corresponded to a single Treponema species, any hits for that species were included as a positive result. Positive and negative predictive values were calculated using microbiome analysis data as the standard for comparison [ 33 ]. Specificity of the qPCR products were verified by a single band at the respective base pair length using 1.4% agarose gel electrophoresis run at 120 V for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Associations between digital dermatitis lesion grades in dairy cattle and the quantities of four Treponema species

Beninger¹,

Naqvi²,

Naushad³

et al. 2018

Vet Res

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Digital dermatitis (DD) presents as painful, ulcerative or proliferative lesions that lead to bovine lameness affecting economic efficiency and animal welfare. Although DD etiological agent(s) have not been established, it is widely accepted that DD is a polymicrobial disease significantly associated with species of Treponema and the non-linear disease progression may be attributed to interactions among infecting bacteria. We postulated the morphological changes associated with DD lesion grades are related to interactions among infecting species of Treponema. We developed a novel species-specific qPCR that can identify the absolute abundance of the four of the most common species of Treponema in DD, T. phagedenis, T. medium, T. pedis and T. denticola, in a single reaction. We found species abundance and the number of distinct Treponema species present is higher in active, ulcerative lesions than in healing lesions, chronic lesions, and DD-free skin. Treponema spp. were present in both DD-free skin and M3 lesions following treatment with oxytetracycline. We have also found positive correlations among T. phagedenis, T. medium and T. pedis indicating they are significantly more likely to be found together than apart and their absolute quantities tend to increase together, a relationship which is not present with T. denticola. Further, we found Treponema, particularly viable T. denticola, in lesions 5 days post treatment with oxytetracycline (M3). Our findings suggest that pathogenicity may be closely associated with Treponema abundance, particularly T. phagedenis, T. medium and T. pedis, and interactions among them, independent of T. denticola. Our results provide a novel, consistent method to identify species of Treponema within DD lesions and associate Treponema spp. and abundance with morphological changes related to host pathogenicity.Electronic supplementary materialThe online version of this article (10.1186/s13567-018-0605-z) contains supplementary material, which is available to authorized users.

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Comparative analysis of the sensitivity of metagenomic sequencing and PCR to detect a biowarfare simulant (Bacillus atrophaeus) in soil samples

Cited by 16 publications

References 47 publications

Development of a New Application for Comprehensive Viability Analysis Based on Microbiome Analysis by Next-Generation Sequencing: Insights into Staphylococcal Carriage in Human Nasal Cavities

Development of a New Application for Comprehensive Viability Analysis Based on Microbiome Analysis by Next-Generation Sequencing: Insights into Staphylococcal Carriage in Human Nasal Cavities

Co-occurrence of Campylobacter Species in Children From Eastern Ethiopia, and Their Association With Environmental Enteric Dysfunction, Diarrhea, and Host Microbiome

Associations between digital dermatitis lesion grades in dairy cattle and the quantities of four Treponema species

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