Exploring the Hospital Microbiome by High-Resolution 16S rRNA Profiling
Abstract
:1. Introduction
2. Results
2.1. Library Characterization
2.2. Composition of Bacterial Communities
2.3. Structure and Diversity of Bacterial Communities
2.4. Single OTUs
2.5. Differential Abundance
2.6. Most Prevalent OTUs Across Samples
2.7. Co-Occurrence Network Analysis
2.8. Pattern of Samples with No Reads
3. Discussion
4. Materials and Methods
4.1. Sampling Site and Collection
4.2. Environment Measurements
4.3. DNA Extraction, PCR Amplification, and Amplicon Sequencing
4.4. 16S rRNA Reads Processing for Downstream Analyses
4.5. Community Composition and Diversity Analysis
4.6. Co-Occurrence Network Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
CDC | Centers for Disease Control and Prevention |
ECU | Emergency Care Unit |
HAIs | Hospital-Acquired Infections |
ICU-A | Intensive Care Unit A |
ICU-B | Intensive Care Unit B |
IU | Inpatient Unit |
MU | Medical Unit |
OTU | Operational Taxonomy Unit |
PCoA | Principal Coordinates Analysis |
SC | Surgery Center |
References
- WHO. Report on the Burden of Endemic Health Care-Associated Infection Worldwide; WHO: Geneva, Switzerland, 2011. [Google Scholar]
- Klevens, R.M.; Edwards, J.R.; Richards, C.L., Jr.; Horan, T.C.; Gaynes, R.P.; Pollock, D.A.; Cardo, D.M. Estimating health care-associated infections and deaths in US hospitals, 2002. Public Health Rep. 2007, 122, 160–166. [Google Scholar] [CrossRef] [PubMed]
- Zimlichman, E.; Henderson, D.; Tamir, O.; Franz, C.; Song, P.; Yamin, C.K.; Keohane, C.; Denham, C.R.; Bates, D.W. Health care-associated infections: A meta-analysis of costs and financial impact on the US health care system. JAMA Intern. Med. 2013, 173, 2039–2046. [Google Scholar] [CrossRef] [PubMed]
- Allegranzi, B.; Nejad, S.B.; Combescure, C.; Graafmans, W.; Attar, H.; Donaldson, L.; Pittet, D. Burden of endemic health-care-associated infection in developing countries: Systematic review and meta-analysis. Lancet 2011, 377, 228–241. [Google Scholar] [CrossRef]
- Weber, D.J.; Anderson, D.; Rutala, W.A. The role of the surface environment in healthcare-associated infections. Curr. Opin. Infect. Dis. 2013, 26, 338–344. [Google Scholar] [CrossRef] [PubMed]
- Dancer, S.J. Controlling hospital-acquired infection: Focus on the role of the environment and new technologies for decontamination. Clin. Microbiol. Rev. 2014, 27, 665–690. [Google Scholar] [CrossRef] [PubMed]
- Suleyman, G.; Alangaden, G.; Bardossy, A.C. The role of environmental contamination in the transmission of nosocomial pathogens and healthcare-associated infections. Curr. Infect. Dis. Rep. 2018, 20, 1–11. [Google Scholar] [CrossRef]
- MacLean, D.; Jones, J.D.; Studholme, D.J. Application of ‘nextgeneration’ sequencing technologies to microbial genetics. Nat. Rev. Microbiol. 2009, 7, 287–296. [Google Scholar] [CrossRef]
- Caporaso, J.G.; Lauber, C.L.; Walters, W.A.; Berg-Lyons, D.; Lozupone, C.A.; Turnbaugh, P.J.; Fierer, N.; Knight, R. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. USA 2011, 108, 4516–4522. [Google Scholar] [CrossRef]
- Gilbert, J.A.; Jansson, J.K.; Knight, R. The Earth Microbiome project: Successes and aspirations. BMC Biol. 2014, 12, 69. [Google Scholar] [CrossRef]
- Thompson, L.R.; The Earth Microbiome Project Consortium; Sanders, J.G.; McDonald, D.; Amir, A.; Ladau, J.; Locey, K.J.; Prill, R.J.; Tripathi, A.; Gibbons, S.M.; et al. A communal catalogue reveals Earth’s multiscale microbial diversity. Nature 2017, 551, 457–463. [Google Scholar] [CrossRef]
- Hilton, S.K.; Castro-Nallar, E.; Pérez-Losada, M.; Toma, I.; McCaffrey, T.A.; Hoffman, E.P.; Siegel, M.O.; Simon, G.L.; Johnson, W.E.; Crandall, K.A. Metataxonomic and metagenomic approaches vs. culture-based techniques for clinical pathology. Front. Microbiol. 2016, 7, 484. [Google Scholar] [CrossRef] [PubMed]
- Padmanabhan, R.; Mishra, A.K.; Raoult, D.; Fournier, P.E. Genomics and metagenomics in medical microbiology. J. Microbiol. Methods 2013, 95, 415–424. [Google Scholar] [CrossRef] [PubMed]
- Fournier, P.E.; Dubourg, G.; Raoult, D. Clinical detection and characterization of bacterial pathogens in the genomics era. Genome Med. 2014, 6, 114. [Google Scholar] [CrossRef] [PubMed]
- Goldberg, B.; Sichtig, H.; Geyer, C.; Ledeboer, N.; Weinstock, G.M. Making the leap from research laboratory to clinic: Challenges and opportunities for next-generation sequencing in infectious disease diagnostics. MBio 2015, 6, e01888-15. [Google Scholar] [CrossRef] [PubMed]
- Poza, M.; Gayoso, C.; Gomez, M.J.; Rumbo-Feal, S.; Tomas, M.; Aranda, J.; Fernandez, A.; Bou, G. Exploring bacterial diversity in hospital environments by GS-FLX Titanium pyrosequencing. PLoS ONE 2012, 7, e44105. [Google Scholar] [CrossRef]
- Hewitt, K.M.; Mannino, F.L.; Gonzalez, A.; Chase, J.H.; Caporaso, J.G.; Knight, R.; Kelley, S.T. Bacterial diversity in two neonatal intensive care units (NICUs). PLoS ONE 2013, 8, e54703. [Google Scholar] [CrossRef] [PubMed]
- Oberauner, L.; Zachow, C.; Lackner, S.; Hogenauer, C.; Smolle, K.H.; Berg, G. The ignored diversity: Complex bacterial communities in intensive care units revealed by 16S pyrosequencing. Sci. Rep. 2013, 3, 1413. [Google Scholar] [CrossRef]
- Tang, C.Y.; Yiu, S.M.; Kuo, H.Y.; Tan, T.S.; Liao, K.H.; Liu, C.C.; Hon, W.K.; Liou, M.L. Application of 16S rRNA metagenomics to analyze bacterial communities at a respiratory care centre in Taiwan. Appl. Microbiol. Biotechnol. 2015, 99, 2871–2881. [Google Scholar] [CrossRef]
- Fadrosh, D.W.; Ma, B.; Gajer, P.; Sengamalay, N.; Ott, S.; Brotman, R.M.; Ravel, J. An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome 2014, 2, 6. [Google Scholar] [CrossRef]
- Castelino, M.; Eyre, S.; Moat, J.; Fox, G.; Martin, P.; Ho, P.; Upton, M.; Barton, A. Optimisation of methods for bacterial skin microbiome investigation: Primer selection and comparison of the 454 versus MiSeq platform. BMC Microbiol. 2017, 17, 23. [Google Scholar] [CrossRef]
- Otter, J.A.; Yezli, S.; French, G.L. The role played by contaminated surfaces in the transmission of nosocomial pathogens. Infect. Control Hosp. Epidemiol. 2011, 32, 687–699. [Google Scholar] [CrossRef] [PubMed]
- Cobrado, L.; Silva-Dias, A.; Azevedo, M.M.; Rodrigues, A.G. High-touch surfaces: Microbial neighbours at hand. Eur. J. Clin. Microbiol. Infect. Dis. 2017, 36, 2053–2062. [Google Scholar] [CrossRef] [PubMed]
- Otter, J.A.; Yezli, S.; Salkeld, J.A.; French, G.L. Evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings. Am. J. Infect. Control 2013, 41, S6–S11. [Google Scholar] [CrossRef] [PubMed]
- Shaughnessy, M.K.; Micielli, R.L.; DePestel, D.D.; Arndt, J.; Strachan, C.L.; Welch, K.B.; Chenoweth, C.E. Evaluation of hospital room assignment and acquisition of Clostridium difficile infection. Infect. Control Hosp. Epidemiol. 2011, 32, 201–206. [Google Scholar] [CrossRef] [PubMed]
- Nseir, S.; Blazejewski, C.; Lubret, R.; Wallet, F.; Courcol, R.; Durocher, A. Risk of acquiring multidrug-resistant Gram-negative bacilli from prior room occupants in the intensive care unit. Clin. Microbiol. Infect. 2011, 17, 1201–1208. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lax, S.; Sangwan, N.; Smith, D.; Larsen, P.; Handley, K.M.; Richardson, M.; Guyton, K.; Krezalek, M.; Shogan, B.D.; Defazio, J.; et al. Bacterial colonization and succession in a newly opened hospital. Sci. Transl. Med. 2017, 9, eaah6500. [Google Scholar] [CrossRef]
- Pereira da Fonseca, T.A.; Pessôa, R.; Felix, A.C.; Sanabani, S.S. Diversity of bacterial communities on four frequently used surfaces in a large Brazilian teaching hospital. Int. J. Environ. Res. Public Health 2016, 13, 152. [Google Scholar] [CrossRef] [PubMed]
- Chen, C.H.; Lin, Y.L.; Chen, K.H.; Chen, W.P.; Chen, Z.F.; Kuo, H.Y.; Hung, H.F.; Tang, C.Y.; Liou, M.L. Bacterial diversity among four healthcare-associated institutes in Taiwan. Sci. Rep. 2017, 7, 8230. [Google Scholar] [CrossRef]
- Dijkshoorn, L. Acinetobacter baumanni. In Molecular Typing in Bacterial Infections; Filippis, I., McKee, M.L., Eds.; Springer: New York, NY, USA, 2013. [Google Scholar]
- Barberán, A.; Bates, S.T.; Casamayor, E.O.; Fierer, N. Using network analysis to explore co-occurrence patterns in soil microbial communities. ISME J. 2012, 6, 343–351. [Google Scholar] [CrossRef]
- Williams, R.J.; Howe, A.; Hofmockel, K.S. Demonstrating microbial co-occurrence pattern analyses within and between ecosystems. Front. Microbiol. 2014, 5, 358. [Google Scholar] [CrossRef]
- Cottee-Jones, H.E.W.; Whittaker, R.J. The keystone species concept: A critical appraisal. Front. Biogeogr. 2012, 4, 117–127. [Google Scholar] [CrossRef]
- Rampelotto, P.H.; Barboza, A.D.; Pereira, A.B.; Triplett, E.W.; Schaefer, C.E.; de Oliveira Camargo, F.A.; Roesch, L.F. Distribution and interaction patterns of bacterial communities in an ornithogenic soil of Seymour Island, Antarctica. Microb. Ecol. 2015, 69, 684–694. [Google Scholar] [CrossRef] [PubMed]
- Gonzalez, D.J.; Haste, N.M.; Hollands, A.; Fleming, T.C.; Hamby, M.; Pogliano, K.; Nizet, V.; Dorrestein, P.C. Microbial competition between Bacillus subtilis and Staphylococcus aureus monitored by imaging mass spectrometry. Microbiology 2011, 157, 2485–2492. [Google Scholar] [CrossRef] [PubMed]
- Bomar, L.; Brugger, S.D.; Yost, B.H.; Davies, S.S.; Lemon, K.P. Corynebacterium accolens releases antipneumococcal free fatty acids from human nostril and skin surface triacylglycerols. MBio 2016, 7, e01725-15. [Google Scholar] [CrossRef] [PubMed]
- Falagas, M.E.; Makris, G.C. Probiotic bacteria and biosurfactants for nosocomial infection control: A hypothesis. J. Hosp. Infect. 2009, 71, 301–306. [Google Scholar] [CrossRef] [PubMed]
- Vandini, A.; Temmerman, R.; Frabetti, A.; Caselli, E.; Antonioli, P.; Balboni, P.G.; Platano, D.; Branchini, A.; Mazzacane, S. Hard surface biocontrol in hospitals using microbial-based cleaning products. PLoS ONE 2014, 9, e108598. [Google Scholar] [CrossRef]
- Caselli, E.; D’Accolti, M.; Vandini, A.; Lanzoni, L.; Camerada, M.T.; Coccagna, M.; Branchini, A.; Antonioli, P.; Balboni, P.G.; Di Luca, D.; et al. Impact of a probiotic-based cleaning intervention on the microbiota ecosystem of the hospital surfaces: Focus on the resistome remodulation. PLoS ONE 2016, 11, e0148857. [Google Scholar] [CrossRef]
- D’Accolti, M.; Soffritti, I.; Mazzacane, S.; Caselli, E. Fighting AMR in the healthcare environment: Microbiome-based sanitation approaches and monitoring tools. Int. J. Mol. Sci. 2019, 20, 1535. [Google Scholar] [CrossRef]
- Caselli, E. Hygiene: Microbial strategies to reduce pathogens and drug resistance in clinical settings. Microb. Biotechnol. 2017, 10, 1079–1083. [Google Scholar] [CrossRef]
- Bock, L.J.; Wand, M.E.; Sutton, J.M. Varying activity of chlorhexidine-based disinfectants against Klebsiella pneumoniae clinical isolates and adapted strains. J. Hosp. Infect. 2016, 93, 42–48. [Google Scholar] [CrossRef]
- Wand, M.E.; Bock, L.J.; Bonney, L.C.; Sutton, J.M. Mechanisms of increased resistance to chlorhexidine and cross-resistance to colistin following exposure of Klebsiella pneumoniae clinical isolates to chlorhexidine. Antimicrob. Agents Chemother. 2017, 61, e01162-16. [Google Scholar] [CrossRef] [PubMed]
- Kembel, S.W.; Jones, E.; Kline, J.; Northcutt, D.; Stenson, J.; Womack, A.M.; Bohannan, B.J.; Brown, G.Z.; Green, J.L. Architectural design influences the diversity and structure of the built environment microbiome. ISME J. 2012, 6, 1469–1479. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Arnold, C. Rethinking sterile. The hospital microbiome. Environ. Health Perspect. 2014, 122, 182–187. [Google Scholar] [CrossRef] [PubMed]
- Al-Ghalith, G.A.; Knights, D. Bygiene: The new paradigm of bidirectional hygiene. Yale J. Biol. Med. 2015, 88, 359–365. [Google Scholar]
- Clarridge, J.E., III. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin. Microbiol, Rev. 2004, 17, 840–862. [Google Scholar] [CrossRef]
- Pei, A.Y.; Oberdorf, W.E.; Nossa, C.W.; Agarwal, A.; Chokshi, P.; Gerz, E.A.; Jin, Z.; Lee, P.; Yang, L.; Poles, M.; et al. Diversity of 16S rRNA genes within individual prokaryotic genomes. Appl. Environ. Microbiol. 2010, 76, 3886–3897. [Google Scholar] [CrossRef] [PubMed]
- Jenkins, C.; Ling, C.L.; Ciesielczuk, H.L.; Lockwood, J.; Hopkins, S.; McHugh, T.D.; Gillespie, S.H. Detection and identification of bacteria in clinical samples by 16S rRNA gene sequencing: Comparison of two different approaches in clinical practice. J. Med. Microbiol. 2012, 61, 483–488. [Google Scholar] [CrossRef]
- Kalia, V.C.; Mukherjee, T.; Bhushan, A.; Joshi, J.; Shankar, P.; Huma, N. Analysis of the unexplored features of rrs (16S rDNA) of the genus Clostridium. BMC Genomics 2011, 12, 18. [Google Scholar] [CrossRef]
- Weigand, M.R.; Pena-Gonzalez, A.; Shirey, T.B.; Broeker, R.G.; Ishaq, M.K.; Konstantinidis, K.T.; Raphael, B.H. Implications of genome-based discrimination between Group I Clostridium botulinum and Clostridium sporogenes strains: Implications for bacterial taxonomy. Appl. Environ. Microb. 2015, 81, 5420–5429. [Google Scholar] [CrossRef]
- Eremeeva, M.E. Molecular Epidemiology of Rickettsial Deseases. In Rickettsiales: Biology, Molecular Biology, Epidemiology, and Vaccine Development (Thomas S); Springer: New York, NY, USA, 2016. [Google Scholar]
- Větrovský, T.; Baldrian, P. The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses. PLoS ONE 2013, 8, e57923. [Google Scholar] [CrossRef]
- Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos, J.; Bealer, K.; Madden, T.L. BLAST+: Architecture and applications. BMC Bioinform. 2009, 10, 421. [Google Scholar] [CrossRef] [PubMed]
- Paulson, J.N.; Stine, O.C.; Bravo, H.C.; Pop, M. Differential abundance analysis for microbial marker-gene surveys. Nat. Methods 2013, 10, 1200–1202. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Caporaso, J.G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F.D.; Costello, E.K.; Fierer, N.; Peña, A.G.; Goodrich, J.K.; I Gordon, J.; et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 2010, 7, 335–336. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Vázquez-Baeza, Y.; Pirrung, M.; Gonzalez, A.; Knight, R. EMPeror: A tool for visualizing high-throughput microbial community data. Gigascience 2013, 2, 16. [Google Scholar] [CrossRef] [PubMed]
- Friedman, J.; Alm, E.J. Inferring correlation networks from genomic survey data. PLoS Comput. Biol. 2012, 8, e1002687. [Google Scholar] [CrossRef] [PubMed]
- Bastian, M.; Heymann, S.; Jacomy, M. Gephi: An open source software for exploring and manipulating networks. AAI Publications. In Proceedings of the Third International AAAI Conference on Weblogs and Social Media, San Jose, CA, USA, 17–20 May 2009. [Google Scholar]
Phyla | Genera | Total | ICU-B | ICU-A | IU | MU | ECU | SC |
---|---|---|---|---|---|---|---|---|
Proteobacteria | Acinetobacter | 17.2 | 17.8 | 15.9 | 18.1 | 14.9 | 21.6 | 15.0 |
Proteobacteria | Pseudomonas | 16.2 | 17.9 | 16.7 | 13.1 | 16.5 | 18.4 | 14.4 |
Firmicutes | Staphylococcus | 6.8 | 9.0 | 8.6 | 6.4 | 5.9 | 3.5 | 7.2 |
Proteobacteria | Klebsiella | 6.6 | 8.5 | 9.0 | 6.5 | 7.2 | 2.2 | 6.4 |
Firmicutes | Streptococcus | 3.5 | 2.3 | 3.8 | 3.5 | 4.5 | 1.4 | 5.7 |
Proteobacteria | Pantoea | 3.4 | 3.5 | 4.9 | 5.0 | 1.0 | 3.4 | 2.9 |
Firmicutes | Bacillus | 3.3 | 1.2 | 2.0 | 3.7 | 5.5 | 6.0 | 1.6 |
Proteobacteria | Escherichia | 2.9 | 4.7 | 3.3 | 2.4 | 2.3 | 2.2 | 2.8 |
Proteobacteria | Stenotrophomonas | 2.3 | 1.7 | 1.7 | 2.7 | 2.8 | 3.6 | 1.5 |
Proteobacteria | Moraxella | 2.2 | 1.7 | 1.7 | 2.5 | 1.7 | 3.1 | 2.5 |
OTU | Species | Bonferroni p | ICU-B | ICU-A | IU | MU | ECU | SC |
---|---|---|---|---|---|---|---|---|
596827 | Pseudomonas stutzeri | 1.78 × 10−10 | 4.41 | 2.34 | 0.55 | 2.92 | 0.51 | 1.20 |
624096 | Acinetobacter baumannii | 7.00 × 10−6 | 4.50 | 4.42 | 1.87 | 3.65 | 1.22 | 3.07 |
623550 | Staphylococcus epidermidis | 2.72 × 10−5 | 4.66 | 4.12 | 3.14 | 3.30 | 1.25 | 4.13 |
602646 | Aerococcus viridans | 0.01 | 0.09 | 0.30 | 0.40 | 0.11 | 0.23 | 1.48 |
617614 | Anaerococcus vaginalis | 0.01 | 1.33 | 0.74 | 0.00 | 0.00 | 0.44 | 0.13 |
587128 | Murdochiella asaccharolytica | 0.01 | 1.30 | 0.57 | 0.00 | 0.00 | 0.34 | 0.24 |
584857 | Porphyromonas bennonis | 0.01 | 1.30 | 0.85 | 0.00 | 0.00 | 0.41 | 0.12 |
624114 | Pseudomonas aeruginosa | 0.01 | 2.35 | 0.99 | 0.48 | 1.52 | 0.32 | 0.92 |
614684 | Acinetobacter lwoffii | 0.01 | 1.85 | 1.15 | 0.97 | 1.17 | 3.23 | 0.35 |
624449 | Escherichia coli | 0.01 | 5.46 | 4.37 | 2.92 | 3.22 | 2.21 | 3.92 |
598572 | Staphylococcus hominis | 0.01 | 2.38 | 1.96 | 1.30 | 0.62 | 0.54 | 0.99 |
607150 | Acinetobacter nosocomialis | 0.02 | 1.36 | 1.02 | 0.00 | 0.00 | 0.66 | 0.25 |
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Rampelotto, P.H.; Sereia, A.F.R.; de Oliveira, L.F.V.; Margis, R. Exploring the Hospital Microbiome by High-Resolution 16S rRNA Profiling. Int. J. Mol. Sci. 2019, 20, 3099. https://doi.org/10.3390/ijms20123099
Rampelotto PH, Sereia AFR, de Oliveira LFV, Margis R. Exploring the Hospital Microbiome by High-Resolution 16S rRNA Profiling. International Journal of Molecular Sciences. 2019; 20(12):3099. https://doi.org/10.3390/ijms20123099
Chicago/Turabian StyleRampelotto, Pabulo H., Aline F.R. Sereia, Luiz Felipe V. de Oliveira, and Rogério Margis. 2019. "Exploring the Hospital Microbiome by High-Resolution 16S rRNA Profiling" International Journal of Molecular Sciences 20, no. 12: 3099. https://doi.org/10.3390/ijms20123099