What Does 16S rRNA Gene-Targeted Next Generation Sequencing Contribute to the Study of Infective Endocarditis in Heart-Valve Tissue?
Abstract
:1. Introduction
2. Results
3. Discussion
4. Materials and Methods
4.1. Samples
4.2. DNA Quantification and Quality Determination
4.3. 16S rRNA Gene Amplification, Library Preparation, and Sequencing
4.4. Sequence Processing and Analysis
5. Conclusions
6. Addendum
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Patient ID | Initial Microbiological Diagnosis before 16S rRNA Gene-Targeted NGS | Identification Technique(s) for Diagnosis | Bacterial Taxa with the Highest Relative Abundance and Minority Findings Suspicious of Being Clinically Important by 16S rRNA Gene-Targeted NGS | |
---|---|---|---|---|
High confident consistent group | #1 | Staphylococcus aureus1 | PCR, WEC 1 | Staphylococcus spp. (99.9%) |
#2 | Staphylococcus epidermidis | BC, PCR | Staphylococcus spp. (99.8%) | |
#3 | Staphylococcus lugdunensis | BC, PCR | Staphylococcus spp. (99.9%) | |
#4 | Streptococcus bovis group | BC, PCR | Streptococcus spp. (99.1%) | |
#5 | S. bovis group | BC, PCR | Streptococcus spp. (99.1%) | |
#6 | Streptococcus milleri | BC | Streptococcus spp. (99.6%) | |
#7 | Streptococcus mitis | BC, PCR | Streptococcus spp. (99.8%) | |
#8 | Streptococus sanguinis | BC, PCR | Streptococcus spp. (99.8%) | |
#9 | S. mitis | BC, PCR | Streptococcus spp. (99.9%) | |
#10 | Streptococcus oralis | BC, PCR | Streptococcus spp. (99.9%) | |
#11 | Coxiella burnetii | PCR | C. burnetii (99.5%) | |
#12 | Enterococcus faecalis | BC, PCR | E. faecalis (99.9%) | |
#13 | E. faecalis | BC, PCR | E. faecalis (99.8%) | |
#14 | E. faecalis | BC, PCR | E. faecalis (99.9%) | |
#15 | E. faecalis | BC, PCR | E. faecalis (99.7%) | |
#16 | Haemophylus parainfluenzae | BC, PCR | H. parainfluenzae (99.5%) | |
#17 | Streptococcus agalactiae | BC, PCR | S. agalactiae (99.9%), Coxiellaceae (<1%) | |
#18 | Streptococcus anginosus group (Streptococcus intermedius 2, S. anginosus 3) | BC 2, PCR 3 | S. anginosus (99.7%), Streptococcus spp. (<1%) | |
Corroborated mixed infections | #19 | Brucella melitensis, C. burnetii4 | BC, PCR, IFA 4, htpAB PCR 4 | Brucellaceae (99.7%), C. burnetii (<1%) |
#20 | E. faecalis, S. epidermidis5 | BC, PCR, CTC 5 | E. faecalis (99.6%), Staphylococcus spp. (<1%) | |
#21 | S. aureus, Escherichia coli6, E. faecalis7 | BC, PCR, VC 6, BC 7† | S. aureus (99.6%), E. faecalis (<1%) | |
Reclassified as mixed infection | #22 | Tropheryma whipplei | PCR | T. whipplei (99.8%), C. burnetii (<1%) |
Low confident consistent group | #23 | E. faecalis | BC, PCR | E. faecalis (68.8%), H. parainfluenzae (29.7%) |
#24 | Streptococcus mutans | BC, PCR | S. mutans (69.5%), E. faecalis (28.6%) | |
New diagnosis | #25 | No etiology | BC, PCR | S. aureus (95.1%) |
Discordant diagnosis | #26 | E. faecalis | BC, PCR | Streptococcus spp. (26.4%), E. faecalis (15.9%) * |
#27 | H. parainfluenzae | BC | Streptococcus spp. (99.2%), H. parainfluenzae (<1%) |
Technique | Advantages | Disadvantages |
---|---|---|
Blood culture | Cornerstone of diagnosis Bacterial identification and susceptibility testing Simple collection procedure Faster if associated with MALDI-TOF Available for clinical microbiology labs | Limited sensitivity, especially after antibiotic therapy or for fastidious microorganisms Delayed diagnosis if negative Processing time: several days |
PCR | More sensitive and faster than culture Applicable for blood and valve tissue (variable collection procedure) Can be broad range or specifically targeted (high specificity) Especially useful for BCNE Processing time: several hours (<1 day) | Variable sensitivity (blood vs. valve; 16S rRNA gene vs. specific targets) Requires careful clinical correlation (detection of viable and non-viable organisms, risk of contamination) Not available for all clinical microbiology labs |
Valve culture | Definitive diagnosis Bacterial identification and susceptibility testing Available for clinical microbiology labs | Low specificity (tedious handling of sample) Limited sensitivity, especially after antibiotic therapy or for fastidious microorganisms Difficulty for sample acquisition (surgery) Delayed diagnosis Processing time: several days |
Serology | Particularly useful in BCNE caused by Coxiella burnetii, Bartonella spp., and other fastidious microorganisms Simple collection procedure Processing time: two hours | Low sensitivity and specificity High seroprevalence in certain collectives for C. burnetii and Bartonella spp. High titers can persist and require careful clinical correlation |
16S rRNA gene-targeted NGS | High-throughput sequencing Detection of all bacteria present in a sample Culture independent Promising diagnostic tool | Variable sensitivity (targeted region, bioinformatics pipeline, equipment, etc.) Lack of consensus for processing and data analysis Bioinformatics skills and computational resources are needed Requires careful clinical correlation (detection of viable and non-viable organisms, risk of contamination) Processing is time-consuming |
ID | Year of Diagnosis | Age, Sex | Affected Tissue | IE Definition * | Cardiac History | Most relevant Historical Conditions | Charlson Comorbidity Index † | Vegetation | Fever | Embolisms | Heart Murmur | Vascular Phenomena | Intracardiac Complications | Cardiac Failure | Antibiotic Therapy (Days) ^ | Mortality |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
#1 | 2012 | 56, F | ivd | P | CD, AVB, PMC | - | 2 | N | Y | N | N | N | N | N | 1 | N |
#2 | 2013 | 69, M | pav | P | CD, AS, MI, HS | - | 3 | Y | Y | N | Y | N | Y | N | 6 | UN |
#3 | 2017 | 67, M | nav | D | CD, AI, AF | ARF | 4 | Y | N | N | N | N | Y | Y | 11 | N |
#4 | 2011 | 64, M | ivd | D | CD, HF, DM, AF | LD | 8 | Y | Y | N | N | N | N | N | 48 | N |
#5 | 2012 | 71, F | nav | D | - | - | 3 | Y | Y | N | Y | N | N | Y | 6 | Y |
#6 | 2014 | 68, F | pmv | D | AF, TI, NVD | n-aHC | 7 | Y | Y | N | N | N | Y | N | 18 | Y |
#7 | 2016 | 53, M | nav | D | - | - | 1 | Y | Y | N | Y | N | Y | N | 8 | N |
#8 | 2016 | 54, F | nmv | D | - | HP | 1 | Y | Y | N | Y | N | N | N | 22 | N |
#9 | 2016 | 53, M | nmv | D | CD | CLD | 2 | Y | N | Y | Y | N | N | N | 18 | N |
#10 | 2017 | 45, M | nmv | D | - | - | 0 | Y | Y | N | Y | N | N | N | 13 | N |
#11 | 2011 | UN, F | ivd | UN | UN | UN | UN | UN | UN | UN | UN | UN | UN | UN | UN | UN |
#12 | 2015 | 69, F | nmv | D | - | GBS | 3 | Y | Y | Y | N | Y | Y | N | 50 | N |
#13 | 2015 | 75, M | nav | D | - | n-aAC | 3 | Y | Y | Y | Y | N | N | N | 17 | N |
#14 | 2015 | 75, M | nmv | D | - | - | 3 | Y | Y | N | Y | N | Y | Y | 22 | N |
#15 | 2017 | 75, M | pav | D | CHF, CD | CLD | 6 | Y | Y | N | Y | N | Y | N | 14 | N |
#16 | 2017 | 26, F | nmv | D | CHD, MI | - | 0 | Y | Y | N | Y | N | N | N | 27 | N |
#17 | 2013 | 45, M | nmv | D | VH, MI | n-aAC, HH | 1 | Y | Y | N | Y | N | N | Y | 15 | Y |
#18 | 2015 | 48, M | nmv | D | MI | - | 0 | Y | Y | Y | N | Y | N | N | 13 | N |
#19 | 2009 | 51, M | ivd | UN | UN | UN | UN | Y | Y | UN | UN | Y | UN | UN | UN | N |
#20 | 2010 | 80, M | nav | D | AF, CD, NVD | n-aPC, ARF | 8 | Y | Y | N | N | N | N | N | 17 | N |
#21 | 2014 | 70, M | nav | D | AF | ITP | 3 | Y | N | N | N | N | Y | Y | 12 | Y |
#22 | 2014 | 48, M | nav | D | - | - | 0 | Y | Y | N | N | N | N | Y | 2 | N |
#23 | 2017 | 84, M | pav | D | - | PVD | 5 | Y | N | N | Y | N | Y | N | 11 | N |
#24 | 2015 | 56, F | nmv | D | NVD | GBD, HYF, FLD | 4 | Y | Y | Y | N | N | Y | N | 11 | N |
#25 | 2011 | 26, M | nmv | D | CHD, VD, WPWS | - | 0 | Y | Y | N | Y | N | Y | N | 5 | N |
#26 | 2013 | 76, M | nmv | D | NVD, AF | COPD, CVDn-aPC | 7 | Y | N | Y | N | N | N | N | 11 | N |
#27 | 2015 | 17, F | ivd | D | CHD | - | 0 | Y | Y | N | N | N | N | N | 12 | N |
Patient ID | Initial Diagnosis | QIIME 1 1 | QIIME 2 2 | Data Refined with BLAST | |||
---|---|---|---|---|---|---|---|
#1 | Staphylococcus aureus | 78.7% | Planococcaceae | 99.9% | Staphylococcus spp. | 99.9% | Staphylococcus spp. |
#2 | Staphylococcus epidermidis | 97.5% | Planococcaceae | 98.4% | Staphylococcus spp. | 99.8% | Staphylococcus spp. |
#3 | Staphylococcus lugdunensis | 97.1% | Planococcaceae | 99.8% | Staphylococcus spp. | 99.9% | Staphylococcus spp. |
#4 | Streptococcus bovis group | 99.9% | Streptoccocus spp. | 99.9% | Streptococcus spp. | 99.1% | Streptococcus spp. |
#5 | S. bovis group | 99.9% | Streptoccocus spp. | 98.6% | Streptoccocus spp. | 99.1% | Streptoccocus spp. |
#6 | Streptococcus milleri | 99.8% | Streptoccocus spp. | 99.3% | Streptoccocus spp. | 99.6% | Streptococcus spp. |
#7 | Streptococcus mitis | 99.9% | Streptoccocus spp. | 99.3% | Streptoccocus spp. | 99.8% | Streptoccocus spp. |
#8 | Streptococcus sanguinis | 99.8% | Streptoccocus spp. | 99.5% | S. sanguinis | 99.8% | Streptoccocus spp. |
#9 | S. mitis | 99.9% | Streptoccocus spp. | 99.7% | Streptoccocus spp. | 99.9% | Streptoccocus spp. |
#10 | Streptococcus oralis | 99.9% | Streptoccocus spp. | 98.8% | Streptoccocus spp. | 99.9% | Streptococcus spp. |
#11 | Coxiella burnetii | 99.5% | Coxiella spp. | 98.8% | C. burnetii | 99.5% | C. burnetii |
#12 | Enteroccus faecalis | 99.4% | Enterococcus spp. | 99.9% | Enterococcus spp. | 99.9% | E. faecalis |
#13 | E. faecalis | 99.1% | Enterococcus spp. | 99.8% | Enterococcus spp. | 99.8% | E. faecalis |
#14 | E. faecalis | 99.5% | Enterococcus spp. | 99.9% | Enterococcus spp. | 99.9% | E. faecalis |
#15 | E. faecalis | 98.8% | Enterococcus spp. | 99.8% | Enterococcus spp. | 99.7% | E. faecalis |
#16 | Haemophilus parainfluenzae | 99.5% | H. parainfluenzae | 88.3% | Haemophilus spp. | 99.5% | H. parainfluenzae |
#17 | Streptococcus agalactiae | 99.9% | Streptoccocus spp. | 97.8% | S. agalactiae | 99.9% | S. agalactiae |
#18 | Streptococcus anginosus group | 99.7% | S. anginosus | 99.6% | S. anginosus subsp. anginosus | 99.7% | S. anginosus |
#19 | Brucella melitensis | 99.7% | Ochrobactrum spp. | 94.7% | Ochrobactrum spp. | 99.7% | Brucellaceae |
#20 | E. faecalis | 99.1% | Enterococcus spp. | 99.6% | Enterococcus spp. | 99.6% | E. faecalis |
#21 | S. aureus | 99.6% | S. aureus | 99.9% | Staphylococcus spp. | 99.6% | S. aureus |
#22 | Tropheryma whipplei | 99.8% | Microbacteriaceae | 98.3% | T. whipplei | 99.8% | T. whipplei |
#23 | E. faecalis | 68.5% | Enterococcus spp. | 62.4% | Enterococcus spp. | 68.8% | E. faecalis |
#24 | Streptococcus mutans | 69.8% | Streptoccocus spp. | 71.1% | S. mutans | 69.5% | S. mutans |
#25 | No etiology | 95.1% | S. aureus | 95.6% | Staphylococcus spp. | 95.1% | S. aureus |
#26 | E. faecalis | 26.9% | Streptoccocus spp. | 25.6% | S. sanguinis | 26.4% | Streptoccocus spp. |
#27 | H. parainfluenzae | 99.8% | Streptoccocus spp. | 55.5% | S. mutans | 99.2% | Streptoccocus spp. |
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Santibáñez, P.; García-García, C.; Portillo, A.; Santibáñez, S.; García-Álvarez, L.; de Toro, M.; Oteo, J.A. What Does 16S rRNA Gene-Targeted Next Generation Sequencing Contribute to the Study of Infective Endocarditis in Heart-Valve Tissue? Pathogens 2022, 11, 34. https://doi.org/10.3390/pathogens11010034
Santibáñez P, García-García C, Portillo A, Santibáñez S, García-Álvarez L, de Toro M, Oteo JA. What Does 16S rRNA Gene-Targeted Next Generation Sequencing Contribute to the Study of Infective Endocarditis in Heart-Valve Tissue? Pathogens. 2022; 11(1):34. https://doi.org/10.3390/pathogens11010034
Chicago/Turabian StyleSantibáñez, Paula, Concepción García-García, Aránzazu Portillo, Sonia Santibáñez, Lara García-Álvarez, María de Toro, and José A. Oteo. 2022. "What Does 16S rRNA Gene-Targeted Next Generation Sequencing Contribute to the Study of Infective Endocarditis in Heart-Valve Tissue?" Pathogens 11, no. 1: 34. https://doi.org/10.3390/pathogens11010034
APA StyleSantibáñez, P., García-García, C., Portillo, A., Santibáñez, S., García-Álvarez, L., de Toro, M., & Oteo, J. A. (2022). What Does 16S rRNA Gene-Targeted Next Generation Sequencing Contribute to the Study of Infective Endocarditis in Heart-Valve Tissue? Pathogens, 11(1), 34. https://doi.org/10.3390/pathogens11010034