Listeria monocytogenes faecal carriage is common and depends on the gut microbiota

AbstractListeria genus comprises two pathogenic species, L. monocytogenes (Lm) and L. ivanovii, and non-pathogenic species. All can thrive as saprophytes, whereas only pathogenic species cause systemic infections. Identifying Listeria species’ respective biotopes is critical to understand the ecological contribution of Listeria virulence. In order to investigate the prevalence and abundance of Listeria species in various sources, we retrieved and analyzed 16S rRNA datasets from MG-RAST metagenomic database. 26% of datasets contain Listeria sensu stricto sequences, and Lm is the most prevalent species, most abundant in soil and host-associated environments, including 5% of human stools. Lm is also detected in 10% of human stool samples from an independent cohort of 900 healthy asymptomatic donors. A specific microbiota signature is associated with Lm faecal carriage, both in humans and experimentally inoculated mice, in which it precedes Lm faecal carriage. These results indicate that Lm faecal carriage is common and depends on the gut microbiota, and suggest that Lm faecal carriage is a crucial yet overlooked consequence of its virulence.

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Listeria monocytogenes faecal carriage is common and driven by microbiota

10.1101/2021.01.13.426560 ◽

2021 ◽

Author(s):

Marc Garcia-Garcera ◽

Lukas Hafner ◽

Christophe Burucoa ◽

Alexandra Moura ◽

Maxime Pichon ◽

...

Keyword(s):

Purifying Selection ◽

Pathogenic Species ◽

Pathogenic Potential ◽

Gut Colonization ◽

Faecal Carriage ◽

Stool Samples ◽

Opportunistic Pathogenic ◽

Systemic Infections ◽

Prevalent Species

AbstractListeria genus comprises two opportunistic pathogenic species, L. monocytogenes (Lm) and L. ivanovii, and several non-pathogenic species. All can thrive as saprophytes, whereas only pathogenic species cause systemic infections in human and cattle. Identifying Listeria species’ respective biotopes is critical to understand the ecological contribution of Listeria pathogenic potential. Here, we aimed at detecting Listeria in samples of diverse origins, to highlight ecological differences between pathogenic and non-pathogenic species. We retrieved 16S rDNA datasets from the metagenomics MG-RAST database and determined the prevalence and abundance of Listeria species in various sources. Overall, Listeria was detected in 14% of datasets. Lm was the most prevalent species, most abundant both in soil and host-associated environments, including in 5% of human stools. Lm was also detected in 10% of human stool samples from an independent cohort of 900 healthy asymptomatic donors. A specific microbiota signature was associated with Lm faecal carriage in human, as well as in experimentally inoculated mice, in which it preceded Lm long-term gut colonization, indicating that gut microbiota composition influences Lm faecal carriage. These results suggest that asymptomatic faecal carriage, rather than disease, exerts purifying selection on Lm “virulence genes”.

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Prevalence and antimicrobial susceptibility of Arcobacter species in human stool samples derived from out- and inpatients: the prospective German Arcobacter prevalence study Arcopath

10.21203/rs.2.19339/v2 ◽

2020 ◽

Author(s):

Vanessa Brückner ◽

Ulrike Fiebiger ◽

Ralf Ignatius ◽

Johannes Friesen ◽

Martin Eisenblätter ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Epidemiological Data ◽

Diagnostic Procedures ◽

Epidemiological Studies ◽

Hospitalized Patients ◽

Prevalence Study ◽

Emerging Pathogens ◽

Stool Samples ◽

Prevalent Species ◽

Human Stool

Abstract Background: Arcobacter species, particularly A. butzleri, but also A. cryaerophilus constitute emerging pathogens causing gastroenteritis in humans. However, isolation of Arcobacter may often fail during routine diagnostic procedures due to the lack of standard protocols. Furthermore, defined breakpoints for the interpretation of antimicrobial susceptibilities of Arcobacter are missing. Hence, reliable epidemiological data of human Arcobacter infections are scarce and lacking for Germany. We therefore performed a 13-month prospective Arcobacter prevalence study in German patients. Results: A total of 4646 human stool samples was included and Arcobacter spp. were identified from 0.85% of specimens in 3884 outpatients and from 0.40% of specimens in 752 hospitalized patients. Overall, A. butzleri was the most prevalent species (n = 24; 67%), followed by A. cryaerophilus (n = 10; 28%) and A. lanthieri (n = 2; 6%). Whereas A. butzleri, A. cryaerophilus and A. lanthieri were identified in outpatients, only A. butzleri could be isolated from samples of hospitalized patients. Antimicrobial susceptibility testing of Arcobacter isolates revealed high susceptibilities to ciprofloxacin, whereas bimodal distributions of MICs were observed for azithromycin and ampicillin.Conclusions: In summary, Arcobacter including A. butzleri, A. cryaerophilus and A. lanthieri could be isolated in 0.85% of German outpatients and ciprofloxacin rather than other antibiotics might be appropriate for antibiotic treatment of infections. Further epidemiological studies are needed, however, to provide a sufficient risk assessment of Arcobacter infections in humans.

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Dead or Alive? Molecular life-dead distinction in human stool samples reveals significantly different composition of the microbial community

10.1101/343194 ◽

2018 ◽

Author(s):

Alexandra Perras ◽

Kaisa Koskinen ◽

Maximilian Mora ◽

Michael Beck ◽

Lisa Wink ◽

...

Keyword(s):

Microbial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Microbial Communities ◽

Human Health ◽

Gene Targeting ◽

Gut Microbiome ◽

Rrna Gene ◽

Stool Samples ◽

Human Stool

AbstractThe gut microbiome is strongly interwoven with human health. Conventional gut microbiome analysis generally involves 16S rRNA gene targeting next generation sequencing (NGS) of stool microbial communities, and correlation of results with clinical parameters. However, some microorganisms may not be alive at the time of sampling, and thus their impact on the human health is potentially less significant. As conventional NGS methods do not differentiate between viable and dead microbial components, retrieved results provide only limited information.Propidium monoazide (PMA) is frequently used in food safety monitoring and other disciplines to discriminate living from dead cells. PMA binds to free DNA and masks it for subsequent procedures. In this article we show the impact of PMA on the results of 16S rRNA gene-targeting NGS from human stool samples and validate the optimal applicable concentration to achieve a reliable detection of the living microbial communities.Fresh stool samples were treated with a concentration series of zero to 300 μM PMA, and were subsequently subjected to amplicon-based NGS. The results indicate that a substantial proportion of the human microbial community is not intact at the time of sampling. PMA treatment significantly reduced the diversity and richness of the sample depending on the concentration and impacted the relative abundance of certain important microorganisms (e.g. Akkermansia, Bacteroides). Overall, we found that a concentration of 100 μM PMA was sufficient to quench signals from disrupted microbial cells.The optimized protocol proposed here can be easily implemented in classical microbiome analyses, and helps to retrieve an improved and less blurry picture of the microbial community composition by excluding signals from background DNA.

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The impact of DNA polymerase and number of rounds of amplification in PCR on 16S rRNA gene sequence data

10.1101/565598 ◽

2019 ◽

Cited By ~ 2

Author(s):

Marc A Sze ◽

Patrick D Schloss

Keyword(s):

16S Rrna ◽

Microbial Communities ◽

Error Rate ◽

Sequence Data ◽

Pcr Amplification ◽

Sequencing Error ◽

Mock Community ◽

Sequencing Error Rate ◽

Stool Samples ◽

Human Stool

AbstractPCR amplification of 16S rRNA genes is a critical, yet under appreciated step in the generation of sequence data to describe the taxonomic composition of microbial communities. Numerous factors in the design of PCR can impact the sequencing error rate, the abundance of chimeric sequences, and the degree to which the fragments in the product represent their abundance in the original sample (i.e. bias). We compared the performance of high fidelity polymerases and varying number of rounds of amplification when amplifying a mock community and human stool samples. Although it was impossible to derive specific recommendations, we did observe general trends. Namely, using a polymerase with the highest possible fidelity and minimizing the number of rounds of PCR reduced the sequencing error rate, fraction of chimeric sequences, and bias. Evidence of bias at the sequence level was subtle and could not be ascribed to the fragments’ fraction of bases that were guanines or cytosines. When analyzing mock community data, the amount that the community deviated from the expected composition increased with rounds of PCR. This bias was inconsistent for human stool samples. Overall the results underscore the difficulty of comparing sequence data that are generated by different PCR protocols. However, the results indicate that the variation in human stool samples is generally larger than that introduced by the choice of polymerase or number of rounds of PCR.ImportanceA steep decline in sequencing costs drove an explosion in studies characterizing microbial communities from diverse environments. Although a significant amount of effort has gone into understanding the error profiles of DNA sequencers, little has been done to understand the downstream effects of the PCR amplification protocol. We quantified the effects of the choice of polymerase and number of PCR cycles on the quality of downstream data. We found that these choices can have a profound impact on the way that a microbial community is represented in the sequence data. The effects are relatively small compared to the variation in human stool samples, however, care should be taken to use polymerases with the highest possible fidelity and to minimize the number of rounds of PCR. These results also underscore that it is not possible to directly compare sequence data generated under different PCR conditions.

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The Impact of DNA Polymerase and Number of Rounds of Amplification in PCR on 16S rRNA Gene Sequence Data

mSphere ◽

10.1128/msphere.00163-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 16

Author(s):

Marc A. Sze ◽

Patrick D. Schloss

Keyword(s):

16S Rrna ◽

Microbial Communities ◽

Error Rate ◽

Sequence Data ◽

Pcr Amplification ◽

Sequencing Error ◽

Mock Community ◽

Sequencing Error Rate ◽

Stool Samples ◽

Human Stool

ABSTRACTPCR amplification of 16S rRNA genes is a critical yet underappreciated step in the generation of sequence data to describe the taxonomic composition of microbial communities. Numerous factors in the design of PCR can impact the sequencing error rate, the abundance of chimeric sequences, and the degree to which the fragments in the product represent their abundance in the original sample (i.e., bias). We compared the performance of high fidelity polymerases and various numbers of rounds of amplification when amplifying a mock community and human stool samples. Although it was impossible to derive specific recommendations, we did observe general trends. Namely, using a polymerase with the highest possible fidelity and minimizing the number of rounds of PCR reduced the sequencing error rate, fraction of chimeric sequences, and bias. Evidence of bias at the sequence level was subtle and could not be ascribed to the fragments’ fraction of bases that were guanines or cytosines. When analyzing mock community data, the amount that the community deviated from the expected composition increased with the number of rounds of PCR. This bias was inconsistent for human stool samples. Overall, the results underscore the difficulty of comparing sequence data that are generated by different PCR protocols. However, the results indicate that the variation in human stool samples is generally larger than that introduced by the choice of polymerase or number of rounds of PCR.IMPORTANCEA steep decline in sequencing costs drove an explosion in studies characterizing microbial communities from diverse environments. Although a significant amount of effort has gone into understanding the error profiles of DNA sequencers, little has been done to understand the downstream effects of the PCR amplification protocol. We quantified the effects of the choice of polymerase and number of PCR cycles on the quality of downstream data. We found that these choices can have a profound impact on the way that a microbial community is represented in the sequence data. The effects are relatively small compared to the variation in human stool samples; however, care should be taken to use polymerases with the highest possible fidelity and to minimize the number of rounds of PCR. These results also underscore that it is not possible to directly compare sequence data generated under different PCR conditions.

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Gut Microbiota Profile in Adult Patients with Idiopathic Nephrotic Syndrome

BioMed Research International ◽

10.1155/2021/8854969 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Hanchang He ◽

Minwa Lin ◽

Lu You ◽

Tongqing Chen ◽

Zijie Liang ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Gut Microbiota ◽

Intestinal Microbiota ◽

Idiopathic Nephrotic Syndrome ◽

Sensu Stricto ◽

Adult Patients ◽

Clinical Parameters ◽

Fecal Microbiome ◽

Nitrogen Levels ◽

Stool Samples

Background. Increasing evidences have reported gut microbiota dysbiosis in many diseases, including chronic kidney disease and pediatric idiopathic nephrotic syndrome (INS). There is lack evidence of intestinal microbiota dysbiosis in adults with INS, however. Here, we to address the association between the gut microbiome and INS. Methods. Stool samples of 35 adult INS patients and 35 healthy volunteers were collected. Total bacterial DNA was extracted, and the V4 regions of the bacterial 16S ribosomal RNA gene were sequenced. The fecal microbiome was analyzed using bioinformatics. The correlation analysis between altered taxa and clinical parameters was also included. Results. We found that microbial diversity in the gut was reduced in adult patients with INS. Acidobacteria, Negativicutes, Selenomonadales, Veillonellaceae, Clostridiaceae, Dialister, Rombousia, Ruminiclostridium, Lachnospira, Alloprevotella, Clostridium sensu stricto, Megamonas, and Phascolarctobacterium were significantly reduced, while Pasteurellales, Parabacteroides, Bilophila, Enterococcus, Eubacterium ventriosum, and Lachnoclostridium were markedly increased in patients with INS. In addition, Burkholderiales, Alcaligenaceae, and Barnesiella were negatively correlated with serum creatinine. Blood urea nitrogen levels were positively correlated with Christensenellaceae, Bacteroidales_S24.7, Ruminococcaceae, Ruminococcus, and Lachnospiraceae_NK4A136, but were negatively correlated with Flavonifractor_plautii and Erysipelatoclostridium_ramosum. Enterobacteriales, Enterobacteriaceae, Porphyromonadaceae, Escherichia/Shigella, Parabacteroides, and Escherichia_coli were positively correlated with albumin. Proteinuria was positively correlated with Verrucomicrobia, Coriobacteriia, Thermoleophilia, Ignavibacteria, Coriobacteriales, Nitrosomonadales, Coriobacteriaceae, and Blautia, but was negatively correlated with Betaproteobacteria, Burkholderiales, and Alcaligenaceae. Conclusion. Our findings show compositional alterations of intestinal microbiota in adult patients with INS and correlations between significantly altered taxa and clinical parameters, which points out the direction for the development of new diagnostics and therapeutic approaches targeted intestinal microbiota.

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Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling

mSystems ◽

10.1128/msystems.00271-18 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 24

Author(s):

Zigui Chen ◽

Pak Chun Hui ◽

Mamie Hui ◽

Yun Kit Yeoh ◽

Po Yee Wong ◽

...

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Rrna Gene ◽

Preservation Solution ◽

Hypervariable Regions ◽

Preservation Solutions ◽

Stool Samples ◽

16S Rrna Gene Primer

ABSTRACT Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservation solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, and Shield) for these aspects. Following storage of human stool samples with these preservatives at room temperature for 7 days, three hypervariable regions of the bacterial 16S rRNA gene (V1-V2, V3-V4, and V4) were amplicon sequenced. We found that samples collected in two preservatives, Norgen and OMNI, showed the least shift in community composition relative to −80°C standards compared with other storage conditions, and both efficiently inhibited the growth of aerobic and anaerobic bacteria. RNAlater did not prevent bacterial activity and exhibited relatively larger community shift. Although the effect of preservation solution was small compared to intersubject variation, notable changes in microbiota composition were observed, which could create biases in downstream data analysis. When community profiles inferred from different 16S rRNA gene hypervariable regions were compared, we found differential sensitivity of primer sets in identifying overall microbial community and certain bacterial taxa. For example, reads generated by the V4 primer pair showed a higher alpha diversity of the gut microbial community. The degenerate 27f-YM primer failed to detect the majority of Bifidobacteriales. Our data indicate that choice of preservation solution and 16S rRNA gene primer pair are critical determinants affecting gut microbiota profiling. IMPORTANCE Large-scale human microbiota studies require specimens collected from multiple sites and/or time points to maximize detection of the small effects in microbe-host interactions. However, batch biases caused by experimental protocols, such as sample collection, massively parallel sequencing, and bioinformatics analyses, remain critical and should be minimized. This work evaluated the effects of preservation solutions and bacterial 16S rRNA gene primer pairs in revealing human gut microbiota composition. Since notable changes in detecting bacterial composition and abundance were observed among choice of preservatives and primer pairs, a consistent methodology is essential in minimizing their effects to facilitate comparisons between data sets.

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Prevalence and antimicrobial susceptibility of Arcobacter species in human stool samples: the prospective German Arcobacter prevalence study Arcopath

10.21203/rs.2.19339/v1 ◽

2019 ◽

Author(s):

Vanessa Brückner ◽

Ulrike Fiebiger ◽

Ralf Ignatius ◽

Johannes Friesen ◽

Martin Eisenblätter ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Epidemiological Data ◽

Diagnostic Procedures ◽

Epidemiological Studies ◽

Hospitalized Patients ◽

Prevalence Study ◽

Emerging Pathogens ◽

Stool Samples ◽

Prevalent Species ◽

Human Stool

Abstract Background Arcobacter species, particularly A. butzleri, but also A. cryaerophilus constitute emerging pathogens causing gastroenteritis in humans. However, isolation of Arcobacter may often fail during routine diagnostic procedures due to the lack of standard protocols. Furthermore, defined breakpoints for the interpretation of antimicrobial susceptibilities of Arcobacter are missing. Hence, reliable epidemiological data of human Arcobacter infections are scarce and lacking for Germany. We therefore performed a 13-month prospective Arcobacter prevalence study in German patients.Results A total of 4646 human stool samples was included and Arcobacter spp. were detected in 0.85% of specimens from 3884 outpatients and in 0.40% of samples from 752 hospitalized patients. Overall, A. butzleri was the most prevalent species (n = 24; 67%), followed by A. cryaerophilus (n = 10; 28%) and A. lanthieri (n = 2; 6%). Whereas A. butzleri, A. cryaerophilus and A. lanthieri were identified in outpatients, only A. butzleri could be isolated from samples of hospitalized patients. Antimicrobial susceptibility testing of Arcobacter isolates revealed high susceptibilities to ciprofloxacin, whereas bimodal distributions of MICs were observed for azithromycin and ampicillin.Conclusions In summary, Arcobacter could be isolated in 0.85% of German outpatients and ciprofloxacin rather than other antibiotics might be appropriate for antibiotic treatment of infections. Further epidemiological studies are needed, however, to provide a sufficient risk assessment of Arcobacter infections in humans.

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Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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Postoperative Complications Are Associated with Long-Term Changes in the Gut Microbiota Following Colorectal Cancer Surgery

Life ◽

10.3390/life11030246 ◽

2021 ◽

Vol 11 (3) ◽

pp. 246

Author(s):

Felix C.F. Schmitt ◽

Martin Schneider ◽

William Mathejczyk ◽

Markus A. Weigand ◽

Jane C. Figueiredo ◽

...

Keyword(s):

Colorectal Cancer ◽

Postoperative Complications ◽

Gut Microbiota ◽

Gut Microbiome ◽

Tumor Resection ◽

Microbial Composition ◽

Colorectal Cancer Surgery ◽

Long Term Changes ◽

Stool Samples

Changes in the gut microbiome have already been associated with postoperative complications in major abdominal surgery. However, it is still unclear whether these changes are transient or a long-lasting effect. Therefore, the aim of this prospective clinical pilot study was to examine long-term changes in the gut microbiota and to correlate these changes with the clinical course of the patient. Methods: In total, stool samples of 62 newly diagnosed colorectal cancer patients undergoing primary tumor resection were analyzed by 16S-rDNA next-generation sequencing. Stool samples were collected preoperatively in order to determine the gut microbiome at baseline as well as at 6, 12, and 24 months thereafter to observe longitudinal changes. Postoperatively, the study patients were separated into two groups—patients who suffered from postoperative complications (n = 30) and those without complication (n = 32). Patients with postoperative complications showed a significantly stronger reduction in the alpha diversity starting 6 months after operation, which does not resolve, even after 24 months. The structure of the microbiome was also significantly altered from baseline at six-month follow-up in patients with complications (p = 0.006). This was associated with a long-lasting decrease of a large number of species in the gut microbiota indicating an impact in the commensal microbiota and a long-lasting increase of Fusobacterium ulcerans. The microbial composition of the gut microbiome shows significant changes in patients with postoperative complications up to 24 months after surgery.

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