Abstract
The muramic acid (MurA) assay is a powerful tool for the detection and quantification of bacteria with no need to enrich samples by culturing. However, the analysis of MurA in mixed biological and environmental matrices is potentially more complex than analysis in isolated bacterial cells. In this study, we employed one commonly used procedure for extraction of MurA from environmental samples and found that the presence of streptomycin interfered with the determination of MurA by creating chemical species that coeluted with the aldononitrile derivative of MurA prepared in this method. On a molar basis, streptomycin yields a signal that is approximately 0.67 times that of MurA. Mass spectrometry analysis confirmed that the interference from hydrolyzed streptomycin is not actually by MurA, but rather is likely to be N-methyl glucosamine. Because streptomycin is widely applied for selective growth of eukaryotes both in situ and in vitro, our findings may have implications for the significance of results from MurA assays. We conclude that MurA remains an effectual bacterial biomarker due to its unique bacterial origin, but care must be applied in interpreting results from the assay when performed in the presence of streptomycin.
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References
Alphei J, Bonkowski M, Scheu S (1995) Application of the selective-inhibition method to determine bacterial–fungal ratios in 3 beechwood soils rich in carbon—optimization of inhibitor concentrations. Biol Fert Soils 19:173–176
Amelung W (2001) Methods using amino sugars as markers for microbial residues in soil. In: Lal R, Kimble JM, Follett RF, Stewart BA (eds) Assessment methods for soil carbon. CRC/Lewis, Boca Raton, pp 233–270
Appuhn A, Joergensen RG, Raubuch M, Scheller E, Wilke B (2004) The automated determination of glucosamine, galactosamine, muramic acid, and mannosamine in soil and root hydrolysates by HPLC. J Plant Nutr Soil Sc 167:17–21
Balkwill DL, Leach FR, Wilson JT, McNabb JF, White DC (1988) Equivalence of microbial biomass measures based on membrane lipid and cell-wall components, adenosine-triphosphate, and direct counts in subsurface aquifer sediments. Microbial Ecol 16:73–84
Benner R, Kaiser K (2003) Abundance of amino sugars and peptidoglycan in marine particulate and dissolved organic matter. Limnol Oceanogr 48:118–128
Fazio SD, Mayberry WR, White DC (1979) Muramic acid assay in sediments. Appl Environ Microb 38:349–350
Findlay RH, Moriarty DJW, White DC (1983) Improved method of determining muramic acid from environmental-samples. Geomicrobiol J 3:135–150
Fox A, Rosario RMT (1994) Quantification of muramic acid, a marker for bacterial peptidoglycan, in dust collected from hospital and home air-conditioning filters using gas chromatography–mass spectrometry. Indoor Air 4:239–247
Gilbart J, Fox A, Whiton RS, Morgan SL (1986) Rhamnose and muramic acid: chemical markers for bacterial cell walls in mammalian tissues. J Microbiol Meth 5:271–282
Glaser B, Turrion MB, Alef K (2004) Amino sugars and muramic acid—biomarkers for soil microbial community structure analysis. Soil Biol Biochem 36:399–407
Guerrant GO, Moss CW (1984) Determination of monosaccharides as aldononitrile, O-methyloxime, alditol, and cyclitol acetate derivatives by gas-chromatography. Anal Chem 56:633–638
Guggenberger G, Frey SD, Six J, Paustian K, Elliott ET (1999) Bacterial and fungal cell-wall residues in conventional and no-tillage agroecosystems. Soil Sci Soc Am J 63:1188–1198
Ingham ER, Coleman DC (1984) Effects of streptomycin, cycloheximide, fungizone, captan, carbofuran, cygon, and PCNB on soil-microorganisms. Microbial Ecol 10:345–358
King JD, White DC (1977) Muramic acid as a measure of microbial biomass in estuarine and marine samples. Appl Environ Microb 33:777–783
Kozar MP, Krahmer MT, Fox A, Larsson L, Allton J (2001) Lunar dust: a negative control for biomarker analyses of extraterrestrial samples. Geochim Cosmochim Ac 65:3307–3317
Liang C, Zhang X, Balser T (2007) Net microbial amino sugar accumulation process in soil as influenced by different plant material inputs. Biol Fert Soils 44:1–7
Millar WN, Casida LE (1970) Evidence for muramic acid in soil. Can J Microbiol 16:299–304
Mimura T, Romano J-C (1985) Muramic acid measurements for bacterial investigations in marine environments by high-pressure liquid chromatography. Appl Environ Microb 50:229–237
Niggemann J, Schubert CJ (2006) Sources and fate of amino sugars in coastal Peruvian sediments. Geochim Cosmochim Ac 70:2229–2237
Rönkkö R, Pennanen T, Smolander A, Kitunen V, Kortemaa H, Haahtela K (1994) Quantification of Frankia strains and other root-associated bacteria in pure cultures and in the rhizosphere of axenic seedlings by high-performance liquid chromatography-based muramic acid assay. Appl Environ Microb 60:3672–3678
Schleife KH, Kandler O (1972) Peptidoglycan types of bacterial cell-walls and their taxonomic implications. Bacteriol Rev 36:407–477
Wang G, He H, Zhang X, Li J, Han L, Wang J (2005) Effects of specific microbial biocides on N transformation in soil with glucose amendment. Chin J Appl Ecol 17:827–830
Whiton RS, Lau P, Morgan SL, Gilbart J, Fox A (1985) Modifications in the alditol acetate method for analysis of muramic acid and other neutral and amino-sugars by capillary gas-chromatography mass-spectrometry with selected ion monitoring. J Chromatogr A 347:109–120
Zhang X, Amelung W (1996) Gas chromatographic determination of muramic acid, glucosamine, mannosamine, and galactosamine in soils. Soil Biol Biochem 28:1201–1206
Acknowledgements
This study was funded by a grant from USDA–CSREES McIntire–Stennis Act. The manuscript has been read and approved by all authors. We wish to thank Dr. Martha Vestling of the Department of Chemistry, University of Wisconsin—Madison, for generously providing us with her advice and expertise.
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Liang, C., Read, H.W. & Balser, T.C. Reliability of Muramic Acid as a Bacterial Biomarker is Influenced by Methodological Artifacts from Streptomycin. Microb Ecol 57, 494–500 (2009). https://doi.org/10.1007/s00248-008-9406-7
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DOI: https://doi.org/10.1007/s00248-008-9406-7