Clinical treatment of the infections caused by various staphylococcal species differ depending on the actual cause of infection. Therefore, it is necessary to develop a fast and reliable method for identification of staphylococci. Raman spectroscopy is an optical method used in multiple scientific fields. Recent studies showed that the method has a potential for use in microbiological research, too. Our work here shows a possibility to identify staphylococci by Raman spectroscopy. We present a method that enables almost 100% successful identification of 16 of the clinically most important staphylococcal species directly from bacterial colonies grown on a Mueller-Hinton agar plate. We obtained characteristic Raman spectra of 277 staphylococcal strains belonging to 16 species from a 24-hour culture of each strain grown on the Mueller-Hinton agar plate using the Raman instrument. The results show that it is possible to distinguish among the tested species using Raman spectroscopy and therefore it has a great potential for use in routine clinical diagnostics.

Department of Microbiology Faculty of Medicine Masaryk University and St Anne's Faculty Hospital Pekařská 53 Brno 65691 Czech Republic

Institute of Scientific Instruments of the Czech Academy of Sciences v v i Královopolská 147 Brno 61264 Czech Republic

National Reference Laboratory for Staphylococci National Institute of Public Health Šrobárova 48 Praha 100 42 Czech Republic

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Vandenbergh MF, Verbrugh HA. Carriage of Staphylococcus aureus: epidemiology and clinical relevance. J. Lab. Clin. Med. 1999;133:525–534. doi: 10.1016/S0022-2143(99)90181-6. PubMed DOI

Piette A, Verschraegen G. Role of coagulase-negative staphylococci in human disease. Vet. Microbiol. 2009;134:45–54. doi: 10.1016/j.vetmic.2008.09.009. PubMed DOI

Kocianova S, et al. Key role of poly-g-DL-glutamic acid in immune evasion and virulence of Staphylococcus epidermidis. J. Clin. Invest. 2005;115:688–694. doi: 10.1172/JCI200523523. PubMed DOI PMC

Oogai Y, Hashimoto M, Kato F, Sugai M, Komatsuzawa H. Expression of Virulence Factors by Staphylococcus aureus Grown in Serum. Appl. Environ. Microbiol. 2011;77:8097–8105. doi: 10.1128/AEM.05316-11. PubMed DOI PMC

Lindberg E, et al. Effect of lifestyle factors on Staphylococcus aureus gut colonization in Swedish and Italian infants. Clin. Microbiol. Infect. 2011;17:1209–1215. doi: 10.1111/j.1469-0691.2010.03426.x. PubMed DOI

van den Berg S, et al. A human monoclonal antibody targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus aureus bacteremia. Int. J. Med. Microbiol. 2015;305:55–64. doi: 10.1016/j.ijmm.2014.11.002. PubMed DOI

Cosgrove SE. The relationship between antimicrobial resistance and patient outcomes: mortality, length of hospital stay, and health care costs. Clin. Infect. Dis. 2006;42(Suppl. 2):S82–S89. doi: 10.1086/499406. PubMed DOI

Kelesidis T, Tsiodras S. Staphylococcus intermedius is not only a zoonotic pathogen, but may also cause skin abscesses in humans after exposure to saliva. Int. J. Infect. Dis. 2010;14:e838–e841. doi: 10.1016/j.ijid.2010.02.2249. PubMed DOI PMC

Durdik P, et al. Staphylococcus intermedius—rare pathogen of acute meningitis. Int. J. Infect. Dis. 2010;14:e236–e238. doi: 10.1016/j.ijid.2009.08.020. PubMed DOI

McCann MT, Gilmore BF, Gorman SP. Staphylococcus epidermidis device-related infections: pathogenesis and clinical management. J. Pharm. Pharmacol. 2008;60:1551–1571. doi: 10.1211/jpp.60.12.0001. PubMed DOI

Jansen B, Hartmann C, Schaumacher-Pedreau F, Peters G. Late onset endopthalmitis associated with intraocular lens: a case of molecularly proved S. epidermidis aetiology. Br. J. Ophthalmol. 1991;75:440–441. doi: 10.1136/bjo.75.7.440. PubMed DOI PMC

Verhoef J, Fleer A. Staphylococcus epidermidis endocarditis and Staphylococcus epidermidis infection in an intensive care unit. Scand. J. Infect. Dis. Suppl. 1983;41:56–64. PubMed

Warren JW. Catheter-associated urinary tract infection. Int. J. Antimicrob. Agents. 2001;17:299–303. doi: 10.1016/S0924-8579(00)00359-9. PubMed DOI

Rupp ME, Archer GL. Coagulase-negative staphylococci: pathogens associated with medical progress. Clin. Infect. Dis. 1994;19:231–245. doi: 10.1093/clinids/19.2.231. PubMed DOI

Rupp ME, Hamer KE. Effect of subinhibitory concentrations of vancomycin, cefazolin, ofloxacin, L-ofloxacin and D-ofloxacin on adherence to intravascular catheters and biofilm formation by Staphylococcus epidermidis. J. Antimicrob. Chemother. 1998;41:155–161. doi: 10.1093/jac/41.2.155. PubMed DOI

Høiby N, et al. ESCMID guideline for the diagnosis and treatment of biofilm infections 2014. Clin. Microbiol. Infect. 2015;21:S1–S25. doi: 10.1016/j.cmi.2014.10.024. PubMed DOI

Nanoukon C, et al. Pathogenic features of clinically significant coagulase-negative staphylococci in hospital and community infections in Benin. Int. J. Med. Microbiol. 2017;307:75–82. doi: 10.1016/j.ijmm.2016.11.001. PubMed DOI

Perdoso SHSP, et al. Biofilm and toxin profile: A phenotypic and genotypic characterization of coagulase-negative staphylococci isolated from human bloodstream infections. Microb. Pathog. 2016;100:312–318. doi: 10.1016/j.micpath.2016.10.005. PubMed DOI

Falcone M, et al. Methicillin-Resistant Staphylococcal Bacteremia in Patients with Hematologic Malignancies: Clinical and Microbiological Retrospective Comparative Analysis of S. haemolyticus, S. epidermidis and S. aureus. J. Chemother. 2004;16:540–548. doi: 10.1179/joc.2004.16.6.540. PubMed DOI

Ertem GT, et al. Peritonitis due to teicoplanin-resistant Staphylococcus haemolyticus. Perit. Dial. Int. 2010;30:117–118. doi: 10.3747/pdi.2008.00274. PubMed DOI

Peel TN, Cole NC, Dylla BL, Patel R. Matrix-assisted laser desorption ionization time of flight mass spectrometry and diagnostic testing for prosthetic joint infection in the clinical microbiology laboratory. Diagn. Microbiol. Infect. Dis. 2015;81:163–168. doi: 10.1016/j.diagmicrobio.2014.11.015. PubMed DOI

Lo DS, Shieh HH, Barreira ER, Ragazzi SL, Gilio AE. High frequency of Staphylococcus saprophyticus urinary tract infections among female adolescents. Pediatr. Infect. Dis. J. 2015;34:1023–1025. doi: 10.1097/INF.0000000000000780. PubMed DOI

Delmas J, et al. Evaluation of the Vitek 2 system with a variety of Staphylococcus species. J. Clin. Microbiol. 2008;46:311–313. doi: 10.1128/JCM.01610-07. PubMed DOI PMC

Marsou R, et al. Distribution of Staphylococcus sciuri subspecies among human clinical specimens, and profile of antibiotic resistance. Res. Microbiol. 1999;150:531–541. doi: 10.1016/S0923-2508(99)00104-7. PubMed DOI

Giordano N, et al. Erythema nodosum associated with Staphylococcus xylosus septicemia. J. Microbiol. Immun. Infect. 2016;49:134–137. doi: 10.1016/j.jmii.2012.10.003. PubMed DOI

Shields BE, Tschetter AJ, Wanat KA. Staphylococcus simulans: An emerging cutaneous pathogen. JAAD Case Rep. 2016;2:428–429. doi: 10.1016/j.jdcr.2016.08.015. PubMed DOI PMC

Pantůček R, et al. Staphylococcus petrasii sp. nov. including S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov., isolated from human clinical specimens and human ear infections. Syst. Appl. Microbiol. 2013;36:90–95. doi: 10.1016/j.syapm.2012.11.004. PubMed DOI

Schie IW, Huser T. Methods and applications of Raman microspectroscopy to single-cell analysis. Appl. Spectrosc. 2013;67:813–828. doi: 10.1366/12-06971. PubMed DOI

Read DS, Whiteley AS. Chemical fixation methods for Raman spectroscopy-based analysis of bacteria. J. Microbiol. Meth. 2015;109:79–83. doi: 10.1016/j.mimet.2014.12.008. PubMed DOI

Maquelin K, et al. Prospective study of the performance of vibrational spectroscopies for rapid identification of bacterial and fungal pathogens recovered from blood cultures. J. Clin. Microbiol. 2013;41:324–329. doi: 10.1128/JCM.41.1.324-329.2003. PubMed DOI PMC

Afseth NK, Bloomfield M, Wold JP, Matousek PA. Novel approach for subsurface through-skin analysis of salmon using spatially offset Raman spectroscopy (SORS) Appl. Spectrosc. 2014;68:255–262. doi: 10.1366/13-07215. PubMed DOI

Notingher I. Raman spectroscopy cell-based biosensors. Sensors. 2007;7:1343–1358. doi: 10.3390/s7081343. DOI

Almarashi JFM, Kapel N, Wilkinson TS, Telle HH. Raman spectroscopy of bacterial species and strains cultivated under reproducible conditions. Spectrosc. Int. J. 2012;27:361–365. doi: 10.1155/2012/540490. DOI

De Gelder J, de Gussem K, Vandenabeele P, Moens L. Reference database of Raman spectra of biological molecules. J. Raman Spectrosc. 2007;38:1133–1147. doi: 10.1002/jrs.1734. DOI

Martinelli A. Effects of a protic ionic liquid on the reaction pathway during non-aqueous sol–gel synthesis of silica: A Raman spectroscopic investigation. Int. J. Mol. Sci. 2014;15:6488–6503. doi: 10.3390/ijms15046488. PubMed DOI PMC

Brauchle E, Schenke-Leyland K. Raman spectroscopy in biomedicine—Non-invasive in vitro analysis of cells and extracellular matrix components in tissues. Biotechnol. J. 2013;8:288–297. doi: 10.1002/biot.201200163. PubMed DOI PMC

Samek O, Al-Marashi JFM, Telle HH. The potential of Raman spectroscopy for the identification of biofilm formation by. Staphylococcus epidermidis. Laser Phys. Lett. 2010;7:378–383. doi: 10.1002/lapl.200910154. DOI

Rebrošová K, et al. (2017) Differentiation between Staphylococcus aureus and Staphylococcus epidermidis strains using Raman spectroscopy. Future Microbiol. 2017;12:881–890. doi: 10.2217/fmb-2016-0224. PubMed DOI

Bernatová S, et al. Following the mechanisms of bacteriostatic versus bactericidal action using Raman spectroscopy. Molecules. 2013;18:13188–13199. doi: 10.3390/molecules181113188. PubMed DOI PMC

Samek O, et al. Raman microspectroscopy of individual algal cells: Sensing unsaturation of storage lipids in vivo. Sensors. 2010;10:8635–8651. doi: 10.3390/s100908635. PubMed DOI PMC

Sandt C, Smith-Palmer T, Pink J, Brennan L, Pink D. Confocal Raman microspectroscopy as a tool for studying the chemical heterogeneities of biofilms in situ. J. Appl. Microbiol. 2007;103:1808–1820. doi: 10.1111/j.1365-2672.2007.03413.x. PubMed DOI

Choo-Smith LP, et al. Investigating microbial (Micro)colony heterogeneity by vibrational spectroscopy. Appl. Environ. Microbiol. 2001;67:1461–1469. doi: 10.1128/AEM.67.4.1461-1469.2001. PubMed DOI PMC

Samek O, et al. Candida parapsilosis Biofilm Identification by Raman Spectroscopy. Int. J. Mol. Sci. 2014;15:23924–23935. doi: 10.3390/ijms151223924. PubMed DOI PMC

Tien NI, et al. Diagnosis of bacterial pathogens in the dialysate of peritoneal dialysis patients with peritonitis using surface-enhanced Raman spectroscopy. Clin. Chim. Acta. 2016;461:69–75. doi: 10.1016/j.cca.2016.07.026. PubMed DOI

Neugebauer U, Rösch P, Popp J. Raman spectroscopy towards clinical application: drug monitoring and pathogen identification. Int. J. Antimicrob. Agents. 2015;46:S35–S39. doi: 10.1016/j.ijantimicag.2015.10.014. PubMed DOI

Kotanen CN, Martinez L, Alvarez J, Simecek JW. Surface enhanced Raman scattering spectroscopy for detection and identification of microbial pathogens isolated from human serum. Sens. Biosensing Res. 2016;8:20–26. doi: 10.1016/j.sbsr.2016.03.002. DOI

Pahlow S, et al. Isolation and identification of bacteria by means of Raman spectroscopy. Adv. Drug Deliv. Rev. 2015;89:105–120. doi: 10.1016/j.addr.2015.04.006. PubMed DOI

Wulf MWH, et al. The use of Raman spectroscopy in the epidemiology of methicillin-resistant Staphylococcus aureus of human- and animal-related clonal lineages. Clin. Microbiol. Infect. 2012;18:147–152. doi: 10.1111/j.1469-0691.2011.03517.x. PubMed DOI

Mathey R, et al. Viability of 3 h grown bacterial micro-colonies after direct Raman identification. J. Microbiol. Methods. 2013;109:67–73. doi: 10.1016/j.mimet.2014.12.002. PubMed DOI

Schuster KC, Urlaub E, Gapes JR. Single-cell analysis of bacteria by Raman microscopy: spectral information on the chemical composition of cells and on the heterogeneity in a culture. J. Microbiol. Methods. 2000;42:29–38. doi: 10.1016/S0167-7012(00)00169-X. PubMed DOI

Mlynáriková K, et al. Influence of Culture Media on Microbial Fingerprints Using Raman Spectroscopy. Sensors. 2015;15:29635–29647. doi: 10.3390/s151129635. PubMed DOI PMC

Liland KH, Almøy T, Mevik BH. Optimal choice of baseline correction for multivariate calibration of spectra. Appl. Spectrosc. 2010;64:1007–1016. doi: 10.1366/000370210792434350. PubMed DOI

Cadusch PJ, Hlaing MM, Wade SA, McArthur SL, Stoddart PR. Improved methods for fluorescence background subtraction from raman spectra. J. Raman. Spectrosc. 2013;44:1587–1595. doi: 10.1002/jrs.4371. DOI

De Luca AC, Mazilu M, Riches A, Herrington CS, Dholakia K. Online fluorescence suppression in modulated raman spectroscopy. Anal. Chem. 2010;82:738–745. doi: 10.1021/ac9026737. PubMed DOI

Brandt NN, Brovko OO, Chikishev AY, Paraschuk OD. Optimization of the rolling-circle filter for raman background subtraction. Appl. Spectrosc. 2006;60:288–293. doi: 10.1366/000370206776342553. PubMed DOI

Fisher RA. The use of multiple measurements in taxonomic problems. Ann. Hum. Genet. 1936;7:179–188.

Sattlecker M, Bessant C, Smith J, Stone N. Investigation of support vector machines and raman spectroscopy for lymph node diagnostics. Analyst. 2010;135:895–901. doi: 10.1039/b920229c. PubMed DOI

Altman NS. An Introduction to Kernel and Nearest-Neighbor Nonparametric Regression. Am. Stat. 1992;46:175–185.

Cortes C, Vapnik V. Support-Vector Networks. Mach. Learn. 1995;20:273–297.

Harz M. Micro-raman spectroscopic identification of bacterial cells of the genus staphylococcus and dependence on their cultivation conditions. Analyst. 2005;130:1543–1550. doi: 10.1039/b507715j. PubMed DOI

Seo Y, Park B, Hinton A, Yoon SC, Lawrence KC. Identification of staphylococcus species with hyperspectral microscope imaging and classification algorithms. Food. Measure. 2016;10:253–263. doi: 10.1007/s11694-015-9301-0. DOI

Allen V, Kalivas JH, Rodriguez RG. Post-consumer plastic identification using raman spectroscopy. Appl Spectrosc. 1999;53:672–681. doi: 10.1366/0003702991947324. DOI

Dingari NC, et al. Development and comparative assessment of raman spectroscopic classification algorithms for lesion discrimination in stereotactic breast biopsies with microcalcifications. J. Biophotonics. 2013;6:371–381. doi: 10.1002/jbio.201200098. PubMed DOI PMC

Verma SP, et al. Resonance Raman spectra of beta-carotene in native and modified low-density lipoprotein. Biochem. Biophys. Res. Commun. 1984;122:867–875. doi: 10.1016/S0006-291X(84)80114-X. PubMed DOI

Fawcett T. An introduction to ROC analysis. Pattern Recogn Lett. 2006;27:861–874. doi: 10.1016/j.patrec.2005.10.010. DOI

Spencer AR, et al. Staphylococcus aureus identification and antibiotic resistance determination using raman spectroscopy. J. Am. Coll. Surgeons. 2011;213:S49. doi: 10.1016/j.jamcollsurg.2011.06.104. DOI

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