scholarly journals The bacterial density of clinical rectal swabs is highly variable, correlates with sequencing contamination, and predicts patient risk of extraintestinal infection

Microbiome ◽  
2022 ◽  
Vol 10 (1) ◽  
Author(s):  
Rishi Chanderraj ◽  
Christopher A. Brown ◽  
Kevin Hinkle ◽  
Nicole Falkowski ◽  
Robert J. Woods ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiome ◽  
Rectal Swab ◽  
Prognostic Significance ◽  
Community Analysis ◽  
Hospitalized Patients ◽  
Bacterial Density ◽  
Skin Bacteria ◽  
Extraintestinal Infection ◽  
Rectal Swabs

Abstract Background In ecology, population density is a key feature of community analysis. Yet in studies of the gut microbiome, bacterial density is rarely reported. Studies of hospitalized patients commonly use rectal swabs for microbiome analysis, yet variation in their bacterial density—and the clinical and methodologic significance of this variation—remains undetermined. We used an ultra-sensitive quantification approach—droplet digital PCR (ddPCR)—to quantify bacterial density in rectal swabs from 118 hospitalized patients. We compared bacterial density with bacterial community composition (via 16S rRNA amplicon sequencing) and clinical data to determine if variation in bacterial density has methodological, clinical, and prognostic significance. Results Bacterial density in rectal swab specimens was highly variable, spanning five orders of magnitude (1.2 × 104–3.2 × 109 16S rRNA gene copies/sample). Low bacterial density was strongly correlated with the detection of sequencing contamination (Spearman ρ = − 0.95, p < 10−16). Low-density rectal swab communities were dominated by peri-rectal skin bacteria and sequencing contaminants (p < 0.01), suggesting that some variation in bacterial density is explained by sampling variation. Yet bacterial density was also associated with important clinical exposures, conditions, and outcomes. Bacterial density was lower among patients who had received piperacillin-tazobactam (p = 0.017) and increased among patients with multiple medical comorbidities (Charlson score, p = 0.0040) and advanced age (p = 0.043). Bacterial density at the time of hospital admission was independently associated with subsequent extraintestinal infection (p = 0.0028), even when controlled for severity of illness and comorbidities. Conclusions The bacterial density of rectal swabs is highly variable, and this variability is of methodological, clinical, and prognostic significance. Microbiome studies using rectal swabs are vulnerable to sequencing contamination and should include appropriate negative sequencing controls. Among hospitalized patients, gut bacterial density is associated with clinical exposures (antibiotics, comorbidities) and independently predicts infection risk. Bacterial density is an important and under-studied feature of gut microbiome community analysis.

scholarly journals Rectal Swabs from Critically Ill Patients Provide Discordant Representations of the Gut Microbiome Compared to Stool Samples

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Katherine Fair ◽  
Daniel G. Dunlap ◽  
Adam Fitch ◽  
Tatiana Bogdanovich ◽  
Barbara Methé ◽  
...  
Keyword(s):  
Critical Illness ◽  
Critically Ill ◽  
Critically Ill Patients ◽  
Gut Microbiome ◽  
Rectal Swab ◽  
Time Points ◽  
Rectal Swabs ◽  
Stool Samples ◽  
Microbiome Profiling

ABSTRACT The role of the gut microbiome in critical illness is being actively investigated, but the optimal sampling methods for sequencing studies of gut microbiota remain unknown. Stool samples are generally considered the reference standard but are not practical to obtain in the intensive care unit (ICU), and thus, rectal swabs are often used. However, the reliability of rectal swabs for gut microbiome profiling has not been established in the ICU setting. In this study, we compared 16S rRNA gene sequencing results between rectal swab and stool samples collected at three time points from mechanically ventilated critically ill adults. Rectal swabs comprised 89% of the samples collected at the baseline time point, but stool samples became more extensively available at later time points. Significant differences in alpha-diversity and beta-diversity between rectal swabs and stool samples were observed, but these differences were primarily due to baseline samples. Higher relative abundances of members of the Actinobacteria phylum (typically skin microbes) were present in rectal swabs than in stool samples (P = 0.05), a difference that was attenuated over time. The progressively increasing similarity of rectal swabs and stool samples likely resulted from increasing levels of stool coating of the rectal vault and direct soiling of the rectal swabs taken at later time points. Therefore, inferences about the role of the gut microbiome in critical illness should be drawn cautiously and should take into account the type and timing of samples analyzed. IMPORTANCE Rectal swabs have been proposed as potential alternatives to stool samples for gut microbiome profiling in outpatients or healthy adults, but their reliability in assessment of critically ill patients has not been defined. Because stool sampling is not practical and often not feasible in the intensive care unit, we performed a detailed comparison of gut microbial sequencing profiles between rectal swabs and stool samples in a longitudinal cohort of critically ill patients. We identified systematic differences in gut microbial profiles between rectal swabs and stool samples and demonstrated that the timing of the rectal swab sampling had a significant impact on sequencing results. Our methodological findings should provide valuable information for the design and interpretation of future investigations of the role of the gut microbiome in critical illness.

scholarly journals Measurement of Klebsiella Intestinal Colonization Density to Assess Infection Risk

2021 ◽  
Author(s):  
Yuang Sun ◽  
Alieysa Patel ◽  
John SantaLucia ◽  
Emily Roberts ◽  
Lili Zhao ◽  
...  
Keyword(s):  
Rectal Swab ◽  
Hospitalized Patients ◽  
Subsequent Infection ◽  
16S Sequencing ◽  
Novel Approach ◽  
Increased Risk ◽  
Rectal Swabs ◽  
Patients At Risk ◽  
Colonization Density ◽  
Highly Correlated

AbstractBackgroundKlebsiella pneumoniae and closely related species K. variicola and K. quasipneumoniae are common causes of healthcare-associated infections, and patients frequently become infected with their intestinal colonizing strain. To assess the association between Klebsiella colonization density and subsequent infections, a case-control study was performed.MethodsA multiplex qPCR assay was developed and validated to quantify Klebsiella (K. pneumoniae, K. variicola, and K. quasipneumoniae combined) relative to total bacterial DNA copies in rectal swabs. Cases of Klebsiella infection were identified based on clinical definitions and having a clinical culture isolate and preceding or co-incident colonization isolate with the same wzi capsular sequence type. Controls were colonized patients without subsequent infection and were matched 2:1 to cases based on age, sex, and rectal swab collection date. Quantitative PCR (qPCR) from rectal swab samples was used to measure the association between relative abundance (RA) of Klebsiella and subsequent infections.ResultsKlebsiella RA by qPCR highly correlated with 16S sequencing (ρ=0.79; P <.001). The median Klebsiella RA in the study group was 2.6% (interquartile range (IQR) 0.1-22.5, n=238), and was higher in cases (15.7%, IQR 0.93-52.6%, n=83) than controls (1.01%, IQR 0.02-12.8%; n=155; P <0.0001). After adjusting for multiple clinical covariates using inverse probability of treatment weighting, subjects with a Klebsiella RA > 22% had a 2.87-fold (1.64-5.03, P =0.0003) increased odds of infection compared to those with lower colonization density levels.ConclusionsMeasurement of colonization density by qPCR could represent a novel approach to identify hospitalized patients at risk for Klebsiella infection.ImportanceColonization by bacterial pathogens often precedes infection, and offers a window of opportunity to prevent these infections. Klebsiella colonization is significantly and reproducibly associated with subsequent infection, however factors that enhance or mitigate this risk in individual patients are unclear. This study developed an assay to measure the density of Klebsiella colonization, relative to total fecal bacteria, in rectal swabs from hospitalized patients. Applying this assay to 238 colonized patients, high Klebsiella density defined as >22% of total bacteria, was significantly associated with subsequent infection. Based on widely available polymerase chain reaction (PCR) technology, this type of assay could be deployed in clinical laboratories to identify patients at increased risk of Klebsiella infections. As novel therapeutics are developed to eliminate pathogens from the gut microbiome, a rapid Klebsiella colonization density assay could identify patients who would benefit from this type of infection prevention interventions.

scholarly journals Diversity and composition of gut microbiome of cervical cancer patients by 16S rRNA and whole-metagenome sequencing

2020 ◽  
Author(s):  
Greyson Biegert ◽  
Tatiana Karpinets ◽  
Xiaogang Wu ◽  
Molly B. El Alam ◽  
Travis T. Sims ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
16S Rrna ◽  
Gut Microbiome ◽  
Rectal Swab ◽  
Gene Sequencing ◽  
National Institutes Of Health ◽  
Cancer Center ◽  
Swab Sample ◽  
Relative Abundances ◽  
Metagenome Sequencing

AbstractPurposeNext generation sequencing has progressed rapidly, characterizing microbial communities beyond culture-based or biochemical techniques. 16S ribosomal RNA gene sequencing (16S) produces reliable taxonomic classifications and relative abundances, while shotgun metagenome sequencing (WMS) allows higher taxonomic and functional resolution at greater cost. The purpose of this study was to determine if 16S and WMS provide congruent information for our patient population from paired fecal microbiome samples.MethodsPatients with locally advanced cervical cancers were enrolled on a prospective, observational clinical trial with a rectal swab sample collected prior to chemoradiation. Bacterial DNA was extracted from each sample and divided in two parts for 16S or WMS sequencing. We used measures of diversity richness and evenness as comparators of 16S and WMS sequencing. Relative abundances of the most common taxa were also compared between both datasets. Both techniques were tested against baseline patient demographics to assess associations identified with either or both methods.ResultsComparative indices were highly congruent between 16S and WMS. The most abundant genera for 16S and WMS data did not overlap. Overlap was observed at the Phylum level, as expected. However, relative abundances correlated poorly between the two methodologies (all p>0.05). Hierarchical clustering of both sequencing analyses identified overlapping enterotypes. Both approaches were in agreement with regard to demographic variables.ConclusionDiversity, evenness and richness are comparable when using 16S and WMS techniques, however relative abundances of individual genera are not. Clinical associations with diversity and evenness metrics were similarly identified with WMS or 16S.ImportanceThe gut microbiome plays an important role in regulating human health and disease. 16S rRNA gene sequencing (16S) and the whole-metagenome shotgun DNA sequencing (WMS) are two approaches to describe the microbial community. 16S sequencing via any amplicon sequencing-based method offers advantages over WMS in terms of precision (specific gene targeting). Additionally, 16S has historically been less costly due to the simplicity of library preparation and it does not require the same level read coverage as WMS. In this study, we performed both sequencing methods on a single rectal swab sample obtained from each cervical cancer patient prior to treatment. We showed that these two methods provide comparable information for diversity, evenness, and richness at higher taxonomic resolution, but are discrepant at a lower resolution. These methodological findings provide valuable information for the design and interpretation of future investigations of the role of the gut microbiome in cancer.Tweet(optional: 256 words, please submit a Tweet that conveys the essential message of your manuscript.) 16S may be sufficient for most initial studies of the gut microbiome in cancer patients, but WMS may be required for analysis of lower level taxonomy.Research supportThis research was supported in part by the Radiological Society of North America Resident/Fellow Award (to L.E.C.), the National Institutes of Health (NIH) through MD Anderson’s Cancer Center Support Grant P30CA016672, the Emerson Collective and the National Institutes of Health T32 grant #5T32 CA101642-14 (T.T.S). This study was partially funded by The University of Texas MD Anderson Cancer Center HPV-related Cancers Moonshot (L.E.C and A.K.).

scholarly journals Prognostic significance of premature ventricular complex burden on hospitalized patients with heart failure

2019 ◽  
Vol 36 (1) ◽  
pp. 134-142
Author(s):  
Shinya Yamada ◽  
Akiomi Yoshihisa ◽  
Takamasa Sato ◽  
Masashi Kamioka ◽  
Takashi Kaneshiro ◽  
...  
Keyword(s):  
Heart Failure ◽  
Prognostic Significance ◽  
Hospitalized Patients ◽  
Premature Ventricular Complex ◽  
Patients With Heart Failure

scholarly journals Comparison of rectal swab, glove tip, and participant-collected stool techniques for gut microbiome sampling

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Meghan I. Short ◽  
Robert Hudson ◽  
Benjamin D. Besasie ◽  
Kelly R. Reveles ◽  
Dimpy P. Shah ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Gut Microbiome ◽  
Rectal Swab ◽  
Prostate Cancer Screening ◽  
San Antonio ◽  
Collection Method ◽  
Gold Standard Method ◽  
Linear Discriminant ◽  
Home Based ◽  
Collection Methods

Abstract Background Studies of the gut microbiome are becoming increasingly important. Such studies require stool collections that can be processed or frozen in a timely manner so as not to alter the microbial content. Due to the logistical difficulties of home-based stool collection, there has been a challenge in selecting the appropriate sample collection technique and comparing results from different microbiome studies. Thus, we compared stool collection and two alternative clinic-based fecal microbiome collection techniques, including a newer glove-based collection method. Results We prospectively enrolled 22 adult men from our prostate cancer screening cohort SABOR (San Antonio Biomarkers of Risk for prostate cancer) in San Antonio, TX, from 8/2018 to 4/2019. A rectal swab and glove tip sample were collected from each participant during a one-time visit to our clinics. A single stool sample was collected at the participant’s home. DNA was isolated from the fecal material and 16 s rRNA sequencing of the V1-V2 and V3-V4 regions was performed. We found the gut microbiome to be similar in richness and evenness, noting no differences in alpha diversity among the collection methods. The stool collection method, which remains the gold-standard method for the gut microbiome, proved to have different community composition compared to swab and glove tip techniques (p< 0.001) as measured by Bray-Curtis and unifrac distances. There were no significant differences in between the swab and glove tip samples with regard to beta diversity (p> 0.05). Despite differences between home-based stool and office-based fecal collection methods, we noted that the distance metrics for the three methods cluster by participant indicating within-person similarities. Additionally, no taxa differed among the methods in a Linear Discriminant Analysis Effect Size (LEfSe) analysis comparing all-against-all sampling methods. Conclusion The glove tip method provides similar gut microbiome results as rectal swab and stool microbiome collection techniques. The addition of a new office-based collection technique could help easy and practical implementation of gut microbiome research studies and clinical practice.

scholarly journals Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2fixation pathway

2017 ◽  
Vol 115 (1) ◽  
pp. E92-E101 ◽  
Author(s):  
Israel A. Figueroa ◽  
Tyler P. Barnum ◽  
Pranav Y. Somasekhar ◽  
Charlotte I. Carlström ◽  
Anna L. Engelbrektson ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
16S Rrna ◽  
Carbon Fixation ◽  
Community Analysis ◽  
Metabolic Model ◽  
Rrna Gene ◽  
Autotrophic Growth ◽  
Anaerobic Wastewater Treatment ◽  
Natural Carbon ◽  
Environmental Relevance

Dissimilatory phosphite oxidation (DPO), a microbial metabolism by which phosphite (HPO32−) is oxidized to phosphate (PO43−), is the most energetically favorable chemotrophic electron-donating process known. Only one DPO organism has been described to date, and little is known about the environmental relevance of this metabolism. In this study, we used 16S rRNA gene community analysis and genome-resolved metagenomics to characterize anaerobic wastewater treatment sludge enrichments performing DPO coupled to CO2reduction. We identified an uncultivated DPO bacterium,CandidatusPhosphitivorax (Ca.P.) anaerolimi strain Phox-21, that belongs to candidate order GW-28 within theDeltaproteobacteria, which has no known cultured isolates. Genes for phosphite oxidation and for CO2reduction to formate were found in the genome ofCa.P. anaerolimi, but it appears to lack any of the known natural carbon fixation pathways. These observations led us to propose a metabolic model for autotrophic growth byCa.P. anaerolimi whereby DPO drives CO2reduction to formate, which is then assimilated into biomass via the reductive glycine pathway.

scholarly journals A pilot study of the effect of deployment on the gut microbiome and traveler’s diarrhea susceptibility

2020 ◽  
Author(s):  
Blake W. Stamps ◽  
Wanda J. Lyon ◽  
Adam P. Irvin ◽  
Nancy Kelley-Loughnane ◽  
Michael S. Goodson
Keyword(s):  
Pilot Study ◽  
16S Rrna ◽  
Gut Microbiome ◽  
16S Rrna Gene Sequencing ◽  
Initial Study ◽  
Mitigation Strategies ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Traveler’S Diarrhea ◽  
Traveler's Diarrhea

AbstractTraveler’s diarrhea (TD) is a recurrent and significant issue for many travelers including the military. While many known enteric pathogens exist that are causative agents of diarrhea, our gut microbiome may also play a role in travelers’ diarrhea susceptibility. To this end we conducted a pilot study of the microbiome of warfighters prior to- and after deployment overseas to identify marker taxa relevant to traveler’s diarrhea. This initial study utilized full-length 16S rRNA gene sequencing to provide additional taxonomic resolution towards identifying predictive taxa.16S rRNA analyses of pre- and post-deployment fecal samples identified multiple marker taxa as significantly differentially abundant in subjects that reported diarrhea, including Weissella, Butyrivibrio, Corynebacterium, uncultivated Erysipelotrichaceae, Jeotgallibaca, unclassified Ktedonobacteriaceae, Leptolinea, and uncultivated Ruminiococcaceae. The ability to identify TD risk prior to travel will inform prevention and mitigation strategies to influence diarrhea susceptibility while traveling.

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