Identifying microbes from environmental water samples
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What is the microbe that we are dealing with? Whether it is cholera or anthrax, we want to know the disease-causing microorganism as quickly as possible since prompt identification of the causative organism would help control disease spread - and potentially save lives through provision of appropriate care and medication. But despite the advent of rapid microbial identification tools – particularly those based on mass spectrometry – most undergraduate curricula continue to focus on culture- and nucleic acid-based identification techniques since they are widely used for detecting and identifying microbes in clinical and environmental samples. Mass spectrometry-based methods, however, have increasingly complemented traditional approaches in clinical and research laboratories - but are rarely featured in undergraduate curricula. Motivated by the desire to address the curriculum gap, I developed an inquirybased laboratory exercise for introducing students to the operating principles and methodology of mass spectrometry-based microbial identification. By requiring students to identify microbes in environmental water samples – a real-life problem with unknown answers – the exercise piqued the students’ interest in learning, while helping to stir their curiosity through an interesting field activity where they put on a scientist’s hat in solving a mystery. This synopsis article summarizes a piece of published educational research and expands on the discussion of concepts underlying matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based microbial identification. For example, the article discusses the relative advantages and disadvantages of the pattern recognition and proteome database search approaches for analyzing mass spectra data. Additionally, the effect of general and tailored sample preparation protocols on identification accuracy is also elaborated. Finally, the pedagogical utility of field- and inquiry-based educational tools is also discussed in greater detail from a post-publication perspective. A full-length synopsis of the work and a structured abstract can be found in the accompanying PDF file, while the original article, “Teaching Microbial Identification with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and Bioinformatics Tools”.
-  Wolk, D. M. & Dunne, W. M. (2011). New Technologies in Clinical Microbiology. Journal of Clinical Microbiology, Vol. 49, pp. S62-S67.
-  Archer, E. & Houldcroft, C. J. (2014). Rabid about whole lyssa genomes. Nature Reviews Microbiology, Vol. 12, pp. 316-316.
-  Bauer, K. A. et al. (2010). An Antimicrobial Stewardship Program's Impact. Clinical Infectious Diseases, Vol. 51, pp. 1074-1080.
-  Hatfull, G. F. (2014). Mycobacteriophages: Windows into Tuberculosis. PLoS Pathogens, doi:10.1371/journal.ppat.1003953.
-  Singh, A. K. et al. (2014). Laser Optical Sensor, a Label-Free On-Plate Salmonella enterica Colony Detection Tool. mBio, doi:10.1128/mBio.01019-13.
-  Kaleta, E. J. et al. (2011). Use of PCR Coupled with Electrospray Ionization Mass Spectrometry for Rapid Identification of Bacterial and Yeast Bloodstream Pathogens from Blood Culture Bottles. Journal of Clinical Microbiology, Vol. 49, pp. 345-353.
-  Ecker, D. J. et al. (2008). Ibis T5000: a universal biosensor approach for microbiology. Nature Reviews Microbiology, Vol. 6, pp. 553-558.
-  Devault, A. M. et al. (2014). Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array. Scientific Reports, doi:10.1038/srep04245.
-  Kloß, S. et al. (2013). Culture Independent Raman Spectroscopic Identification of Urinary Tract Infection Pathogens: A Proof of Principle Study. Analytical Chemistry, Vol. 85, pp. 96109616.
-  Safari, M., Amache, R., Esmaeilishirazifard, E. & Keshavarz, T. (2014). Microbial metabolism of quorum-sensing molecules acyl-homoserine lactones, γ-heptalactone and other lactones. Applied Microbiology and Biotechnology, Vol. 98, pp. 3401-3412.
-  Li, X.-R., Lv, Y., Meng, H., Gu, J.-D. & Quan, Z.-X. (2014). Analysis of microbial diversity by pyrosequencing the small-subunit ribosomal RNA without PCR amplification. Applied Microbiology and Biotechnology, Vol. 98, pp. 3777-3789.
-  Vital, M., Howe, A. C. & Tiedje, J. M. (2014), Revealing the Bacterial Butyrate Synthesis Pathways by Analyzing (Meta)genomic Data. mBio, doi:10.1128/mBio.00889-14.
-  Park, S. J. et al. (2014). Detection of microorganisms using terahertz metamaterials. Scientific Reports, doi:10.1038/srep04988.
-  Lleo, M.M. et al. (2014). Detecting the presence of bacterial DNA by PCR can be useful in diagnosing culture-negative cases of infection, especially in patients with suspected infection and antibiotic therapy. FEMS Microbiology Letters, Vol. 354, pp. 153-160.
-  Sandrin, T. R. & Demirev, P. A. (2014). Using Mass Spectrometry to Identify and Characterise Bacteria. Microbe, Vol. 9, pp. 23-29.
-  Mahé, P. et al. (2014). Automatic identification of mixed bacterial species fingerprints in a MALDI-TOF mass-spectrum. Bioinformatics, doi:10.1093/bioinformatics/btu022.
-  Seng, P. et al. (2009), Ongoing Revolution in Bacteriology: Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry. Clinical Infectious Diseases, Vol. 49, pp. 543-551.
-  Chen, J. H. K. et al. (2013). Direct Bacterial Identification in Positive Blood Cultures using two commercial MALDI-TOF mass spectrometry systems. Journal of Clinical Microbiology, doi:10.1128/jcm.03259-12.
-  Steensels, D., Verhaegen, J. & Lagrou, K. (2011). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of bacteria and yeasts in a clinical microbiological laboratory: A review. Acta Clinica Belgica, Vol. 66, pp. 267-273.
-  Ng, W. (2013). Teaching Microbial Identification with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and Bioinformatics Tools. Journal of Microbiology and Biology Education, Vol. 14, pp. 103106.
-  Marko, D. C. et al. (2012). Evaluation of the Bruker Biotyper and Vitek MS MatrixAssisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems for Identification of Nonfermenting Gram-Negative Bacilli Isolated from Cultures from Cystic Fibrosis Patients. Journal of Clinical Microbiology, Vol. 50, pp. 2034-2039.
-  Anhalt, J. P. & Fenselau, C. (1975). Identification of bacteria using mass spectrometry. Analytical Chemistry, Vol. 47, pp. 219-225.
-  Barbuddhe, S. B. et al. (2008). Rapid Identification and Typing of Listeria Species by MatrixAssisted Laser Desorption Ionization-Time of Flight Mass Spectrometry. Applied and Environmental Microbiology, Vol. 74, pp. 5402-5407.
-  Murray, P. R. (2012). What Is New in Clinical Microbiology—Microbial Identification by MALDI-TOF Mass Spectrometry: A Paper from the 2011 William Beaumont Hospital Symposium on Molecular Pathology. The Journal of molecular diagnostics: JMD, Vol. 14, pp. 419-423.
-  van Veen, S. Q., Claas, E. C. J. & Kuijper, E. J. (2010). High-Throughput Identification of Bacteria and Yeast by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Conventional Medical Microbiology Laboratories. Journal of Clinical Microbiology, Vol. 48, pp. 900-907.
-  Usbeck, J., Wilde, C., Bertrand, D., Behr, J. & Vogel, R. (2014). Wine yeast typing by MALDITOF MS. Applied Microbiology and Biotechnology, Vol. 98, pp. 3737-3752.
-  Mellmann, A. et al. (2008). Evaluation of Matrix-Assisted Laser Desorption IonizationTimeof-Flight Mass Spectrometry in Comparison to 16S rRNA Gene Sequencing for Species Identification of Nonfermenting Bacteria. Journal of Clinical Microbiology, Vol. 46, pp. 19461954.
-  Eddabra, R., Prévost, G. & Scheftel, J.-M. (2012). Rapid discrimination of environmental Vibrio by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Microbiological Research, Vol. 167, pp. 226-230.
-  Horká, M. et al. (2013). CIEF separation of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line MALDI-TOF MS identification. Analytica Chimica Acta, Vol. 788, pp. 193-199.
-  Šedo, O., Vávrová, A., Vad'urová, M., Tvrzová, L. & Zdráhal, Z. (2013). The influence of growth conditions on strain differentiation within the Lactobacillus acidophilus group using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling. Rapid Communications in Mass Spectrometry, Vol. 27, pp. 2729-2736.
-  Abdelhamid, H. N., Khan, M. S. & Wu, H.-F. (2014). Design, characterization and applications of new ionic liquid matrices for multifunctional analysis of biomolecules: A novel strategy for pathogenic bacteria biosensing. Analytica Chimica Acta, Vol. 823, pp. 51-60. DOI:10.1016/j.aca.2014.03.026
-  Dybwad, M., van der Laaken, A. L., Blatny, J. M. & Paauw, A. (2013). Rapid Identification of Bacillus anthracis Spores in Suspicious Powder Samples by Using Matrix-Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry (MALDI-TOF MS). Applied and Environmental Microbiology, Vol. 79, pp. 5372-5383.
-  El-Bouri, K. et al. (2012). Comparison of bacterial identification by MALDI-TOF mass spectrometry and conventional diagnostic microbiology methods: agreement, speed and cost implications. British Journal of Biomedical Science, Vol. 69, pp. 47-55.
-  Biswas, S. & Rolain, J.-M. (2013). Use of MALDI-TOF mass spectrometry for identification of bacteria that are difficult to culture. Journal of Microbiological Methods, Vol. 92, pp. 14-24.
-  Ziegler, D. et al. (2012). In Situ Identification of Plant-Invasive Bacteria with MALDITOF Mass Spectrometry. PLoS ONE, doi:10.1371/journal.pone.0037189.
-  Wahl, K.L. et al. (2002). Analysis of Microbial Mixtures by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Analytical Chemistry, Vol. 74, pp. 6191-6199.
-  Edwards-Jones, V. et al. (2000). Rapid discrimination between methicillin-sensitive and methicillin-resistant Staphylococcus aureus by intact cell mass spectrometry. Journal of Medical Microbiology, Vol. 49, pp. 295-300.
-  Zongmin Du, Ruifu Yang, Zhaobiao Guo, Yajun Song, and Jin Wang (2002). Identification of Staphylococcus aureus and determination of its methicillin resistance by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry. Analytical Chemistry, Vol. 74, pp. 5487-5491.
-  Balážová, T. et al. (2014). The influence of culture conditions on the identification of Mycobacterium species by MALDI-TOF MS profiling. FEMS Microbiology Letters, Vol. 353, pp. 77-84.
-  Fagerquist, C. K. et al. (2014). Top-Down Proteomic Identification of Shiga Toxin 2 Subtypes from Shiga Toxin-Producing Escherichia coli by Matrix-Assisted Laser Desorption Ionization–Tandem Time of Flight Mass Spectrometry. Applied and Environmental Microbiology, Vol. 80, pp. 2928-2940.
-  Chubukov, V., Gerosa, L., Kochanowski, K. & Sauer, U. (2014). Coordination of microbial metabolism. Nature Reviews Microbiology, Vol. 12, pp. 327-340.
-  Jarman, K.H. et al. (2000), An Algorithm for Automated Bacterial Identification Using Matrix Assisted Laser Desorption/Ionization Mass Spectrometry. Analytical Chemistry, Vol. 72, pp. 1217-1223.
-  Wynne, C., Fenselau, C., Demirev, P. A. & Edwards, N. (2009). Top-Down Identification of Protein Biomarkers in Bacteria with Unsequenced Genomes. Analytical Chemistry, Vol. 81, pp. 9633-9642.
-  Demirev, P. A., Ho, Y.-P., Ryzhov, V. & Fenselau, C. (1999). Microorganism Identification by Mass Spectrometry and Protein Database Searches. Analytical Chemistry, Vol. 71, pp. 27322738.
-  Nie, Y. et al. (2014). Diverse alkane hydroxylase genes in microorganisms and environments. Scientific Reports, doi:10.1038/srep04968.
-  Bohlin, J., Sekse, C., Skjerve, E. & Brynildsrud, O. (2014). Positive correlations between genomic % AT and genome size within strains of bacterial species. Environmental Microbiology Reports, Vol. 6, pp. 278-286.
-  Bryant, T. N. (1994). A bacterial identification teaching exercise revisited. Computer applications in the biosciences: CABIOS, Vol. 10, pp. 329-334.
-  Vanchieri, C. (2013). Partners for the environment. HHMI Bulletin, Vol. Fall 2013, pp. 34-35.
-  Drissner, J. R., Haase, H.-M., Wittig, S. & Hille, K. (2013). Short-term environmental education: long-term effectiveness? Journal of Biological Education, Vol. 48, pp. 9-15.
-  Dopico, E., Linde, A.R. & Garcia-Vazquez, E. (2013). Learning gains in lab practices: teach science doing science. Journal of Biological Education, Vol. 48, pp. 46-52.
-  Pienta, N. J. (2014). Teaching General Chemistry and Making a Difference. Journal of Chemical Education, Vol. 91, pp. 305-306.
-  Conway, C. J. (2014). Effects of Guided Inquiry versus Lecture Instruction on Final Grade Distribution in a One-Semester Organic and Biochemistry Course. Journal of Chemical Education, Vol. 91, pp. 480-483.
-  Mandler, D., Blonder, R., Yayon, M., Mamlok-Naaman, R. & Hofstein, A. (2014). Developing and Implementing Inquiry-Based, Water Quality Laboratory Experiments for High School Students To Explore Real Environmental Issues Using Analytical Chemistry. Journal of Chemical Education, Vol. 91, pp. 492-496.
-  Eisen, L., Marano, N. & Glazier, S. (2014). Activity-Based Approach For Teaching Aqueous Solubility, Energy, and Entropy. Journal of Chemical Education, Vol. 91, pp. 484-491.
-  Jordan, T. C. et al. (2014). A Broadly Implementable Research Course in Phage Discovery and Genomics for First-Year Undergraduate Students. mBio, doi:10.1128/mBio.01051-13.
-  Linn, M. C. et al. (2014). Computer-Guided Inquiry to Improve Science Learning. Science, Vol. 344, pp. 155-156.
-  Fakayode, S. O., Yakubu, M., Adeyeye, O. M., Pollard, D. A. & Mohammed, A. K. (2014), Promoting Undergraduate STEM Education at a Historically Black College and University through Research Experience. Journal of Chemical Education, Vol. 91, pp. 662-665.
-  Khatib, F. et al. (2011). Crystal structure of a monomeric retroviral protease solved by protein folding game players. Nature Structural & Molecular Biology, Vol. 18, pp. 11751177.
-  Müller, T. & Ruppel, S. (2014). Progress in cultivation-independent phyllosphere microbiology. FEMS Microbiology Ecology, Vol. 87, pp. 2-17.
-  Romero, F. M., Marina, M. & Pieckenstain, F. L. (2014). The communities of tomato (Solanum lycopersicum L.) leaf endophytic bacteria, analyzed by 16S-ribosomal RNA gene pyrosequencing. FEMS Microbiology Letters, Vol. 351, pp. 187-194.
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