Skip to main content
SpringerLink
Log in

Fc-MBL-modified Fe3O4 magnetic bead enrichment and fixation in Gram stain for rapid detection of low-concentration bacteria

  • Original Paper
  • Published:
Microchimica Acta Aims and scope Submit manuscript

Abstract

Functional bacterial enrichment magnetic beads (Fe3O4@SiO2@Fc-MBL) and Gram staining were combined for the fast diagnosis of infecting bacteria in meningitis. Fe3O4@SiO2@Fc-MBL has excellent microbial binding ability and can be used for bacterial enrichment from cerebrospinal fluid (CSF). The enriched bacteria are recognized by Gram stain at very low concentrations (10 CFU·mL−1). The feasibility of this method was verified by five common bacteria in meningitis infection (Gram-positive: Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus capitis; Gram-negative: Klebsiella pneumoniae and Escherichia coli). The extraction efficiency of Fc-MBL-modified Fe3O4 magnetic beads was approximately 90% in artificial CSF for the selected bacteria, with the exception of E. coli (~ 60%). The bacteria were successfully recognized by Gram staining and microscopic observation. Fe3O4@SiO2@Fc-MBL acts by capturing and fixing the bacteria in a magnetic field throughout the experiment. Compared with traditional CSF Gram staining, this new method avoids interference by inflammatory cells and red blood cells during microscopic examination. Furthermore, the sensitivity of this method is much better than the centrifugation smear method. The whole process can be accomplished within 30 min. This novel method may have potential as a clinical tool for analysis of bacteria in the CSF.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Kim KS (2010) Acute bacterial meningitis in infants and children. Lancet Infect Dis 10(1):32–42. https://doi.org/10.1016/S1473-3099(09)70306-8

    Article  CAS  PubMed  Google Scholar 

  2. Kim BJ, Shusta EV, Doran KS (2019) Past and current perspectives in modeling bacteria and blood brain barrier interactions. Front Microbiol 10:1336. https://doi.org/10.3389/fmicb.2019.01336

    Article  PubMed  PubMed Central  Google Scholar 

  3. Guo LY, Zhang ZX, Wang X, Zhang PP, Shi W, Yao KH, Liu LL, Liu G, Yang YH (2016) Clinical and pathogenic analysis of 507 children with bacterial meningitis in Beijing, 2010–2014. Int J Infect Dis 50:38–43. https://doi.org/10.1016/j.ijid.2016.07.010

    Article  PubMed  Google Scholar 

  4. Mwenda JM, Soda E, Weldegebriel G, Katsande R, Biey JNM, Traore T, de Gouveia L, du Plessis M, von Gottberg A, Antonio M, Kwambana-Adams B, Worwui A, Gierke R, Schwartz S, Van Beneden C, Cohen A, Serhan F, Lessa FC (2019) Pediatric bacterial meningitis surveillance in the World Health Organization African Region using the Invasive Bacterial Vaccine-Preventable Disease Surveillance Network. 2011–2016 Clin Infect Dis 69: S49-S57. https://doi.org/10.1093/cid/ciz472

  5. Yang MX, Ault MJ (2021) Improving speed of cerebrospinal fluid collection safely during lumbar punctures. Ann Emerg Med 77(1):129–130. https://doi.org/10.1016/j.annemergmed.2020.07.001

    Article  PubMed  Google Scholar 

  6. Lyons TW, Cruz AT, Freedman SB, Neuman MI, Balamuth F, Mistry RD, Mahajan P, Aronson PL, Thomson JE, Pruitt CM, Nigrovic LE (2017) Interpretation of cerebrospinal fluid white blood cell counts in young infants with a traumatic lumbar puncture. Ann Emerg Med 69(5):622–631. https://doi.org/10.1016/j.annemergmed.2016.10.008

    Article  PubMed  Google Scholar 

  7. Fitch MT, van de Beek D (2007) Emergency diagnosis and treatment of adult meningitis. Lancet Infect Dis 7(3):191–200. https://doi.org/10.1016/S1473-3099(07)70050-6

    Article  PubMed  Google Scholar 

  8. Hasbun R, Bijlsma M, Brouwer MC, Khoury N, Hadi CM, van der Ende A, Wootton SH, Salazar L, Hossain MM, Beilke M, van de Beek D (2013) Risk score for identifying adults with CSF pleocytosis and negative CSF Gram stain at low risk for an urgent treatable cause. J Infection 67(2):1–9. https://doi.org/10.1016/j.jinf.2013.04.002

    Article  Google Scholar 

  9. Brouwer MC, Thwaites GE, Tunkel AR, van de Beek D (2012) Dilemmas in the diagnosis of acute community-acquired bacterial meningitis. Lancet 380(9854):1684–1692. https://doi.org/10.1016/S0140-6736(12)61185-4

    Article  PubMed  Google Scholar 

  10. Wagner K, Springer B, Pires VP, Keller PM (2018) Pathogen identification by multiplex LightMix real-time PCR assay in patients with meningitis and culture-negative cerebrospinal fluid specimens. J Clin Microbiol 56(2):e01492-e1517. https://doi.org/10.1128/JCM.01492-17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Li H, Li LL, Chi YY, Tian QW, Zhou TT, Han CH, Zhu YQ, Zhou YS (2020) Development of a standardized Gram stain procedure for bacteria and inflammatory cells using an automated staining instrument. Microbiologyopen 9(9):e1099. https://doi.org/10.1002/mbo3.1099

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Miller S, Naccache SN, Samayoa E, Messacar K, Arevalo S, Federman S, Stryke D, Pham E, Fung B, Bolosky WJ, Ingebrigtsen D, Lorizio W, Paff SM, Leake JA, Pesano R, DeBiasi R, Dominguez S, Chiu CY (2019) Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid. Genome Res 29(5):831–842. https://doi.org/10.1101/gr.238170.118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wilson MR, Sample HA, Zorn KC, Arevalo S, Yu G, Neuhaus J, Federman S, Stryke D, Briggs B, Langelier C, Berger A, Douglas V, Josephson SA, Chow FC, Fulton BD, DeRisi JL, Gelfand JM, Naccache SN, Bender J, Bard JD, Murkey J, Carlson M, Vespa PM, Vijayan T, Allyn PR, Campeau S, Humphries RM, Klausner JD, Ganzon CD, Memar F, Ocampo NA, Zimmermann LL, Cohen SH, Polage CR, DeBiasi RL, Haller B, Dallas R, Maron G, Hayden R, Messacar K, Dominguez SR, Miller S, Chiu CY (2019) Clinical metagenomic sequencing for diagnosis of meningitis and encephalitis. New Engl J Med 380(24):2327–2340. https://doi.org/10.1056/NEJMoa1803396

    Article  CAS  PubMed  Google Scholar 

  14. Thomson RB (2016) One small step for the Gram stain, one giant leap for clinical microbiology. J Clin Microbiol 54(6):1416–1417. https://doi.org/10.1128/JCM.00303-16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Guarner J, Street C, Matlock M, Cole L, Brierre F (2017) Improving Gram stain proficiency in hospital and satellite laboratories that do not have microbiology. Clin Chem Lab Med 55(3):458–461. https://doi.org/10.1515/cclm-2016-0556

    Article  CAS  PubMed  Google Scholar 

  16. Halladin DK, Ortega FE, Ng KM, Footer MJ, Mitic NS, Malkov SN, Gopinathan A, Huang KC, Theriot JA (2021) Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall. Nat Microbiol 6(8):1055. https://doi.org/10.1101/2020.11.24.396366

    Article  CAS  PubMed  Google Scholar 

  17. Forster S, Snape JR, Lappin-Scott HM, Porter J (2002) Simultaneous fluorescent gram staining and activity assessment of activated sludge bacteria. Appl Environ Microb 68(10):4772–4779. https://doi.org/10.1128/AEM.68.10.4772-4779.2002

    Article  CAS  Google Scholar 

  18. Sharma S, Acharya J, Banjara MR, Ghimire P, Singh A (2020) Comparison of acridine orange fuorescent microscopy and gram stain light microscopy for the rapid detection of bacteria in cerebrospinal fuid. BMC Res Notes 13(1):29. https://doi.org/10.1186/s13104-020-4895-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Neeraja M, Lakshmi V, Padmasri C, Padmaja KJ (2017) Utility of Acridine Orange staining for detection of bacteria from positive blood cultures. Microbiol Meth 139:215–217. https://doi.org/10.1016/j.mimet.2017.06.014

    Article  CAS  Google Scholar 

  20. Baron EJ, Mix S, Moradi WJ (2010) Clinical utility of an automated instrument for Gram staining single slides. Clin Microbiol 48(6):2014–2015. https://doi.org/10.1128/JCM.02503-09

    Article  Google Scholar 

  21. Lambrecht J, Cichocki N, Hubschmann T, Koch C, Harms H, Muller S (2017) Flow cytometric quantifcation, sorting and sequencing of methanogenic archaea based on F420 autofuorescence. Microb Cell Fact 16:180. https://doi.org/10.1186/s12934-017-0793-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kim SY, Park Y, Kim H, Kim J, Koo SH, Kwon GC (2018) Rapid screening of urinary tract infection and discrimination of Gram-positive and Gram-negative bacteria by automated flow cytometric analysis using Sysmex UF-5000. J Clin Microbiol 56(8):e02004-e2017. https://doi.org/10.1128/JCM.02004-17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Shanholtzer CJ, Schaper PJ, Peterson LR (1982) Concentrated gram stain smears prepared with a cytospin centrifuge. J Clin Microbiol 16(6):1052–1056. https://doi.org/10.1128/JCM.16.6.1052-1056.1982

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Guo RY, Huang FC, Cai GZ, Zheng LY, Xue L, Li YB, Liao M, Wang MH, Lin JH (2020) A colorimetric immunosensor for determination of foodborne bacteria using rotating immunomagnetic separation, gold nanorod indication, and click chemistry amplification. Microchim Acta 187(4):197. https://doi.org/10.1007/s00604-020-4169-z

    Article  CAS  Google Scholar 

  25. Yazdankhah SP, Sorum H, Larsen HJS, Gogstad G (2001) Use of magnetic beads for Gram staining of bacteria in aqueous suspension. J Microbiol Meth 47(3):369–371. https://doi.org/10.1016/S0167-7012(01)00350-5

    Article  CAS  Google Scholar 

  26. Budin G, Chung HJ, Lee H, Weissleder R (2012) A magnetic Gram stain for bacterial detection. Angew Chem Int Edit 51(31):7752–7755. https://doi.org/10.1002/anie.201202982

    Article  CAS  Google Scholar 

  27. Silva PMD, de Oliveira WF, Albuquerque PBS, Correia MTD, Coelho LCBB (2019) Insights into anti-pathogenic activities of mannose lectins. Int J Biol Macromol 140:234–244. https://doi.org/10.1016/j.ijbiomac.2019.08.059

    Article  CAS  Google Scholar 

  28. Gingras AR, Girija UV, Keeble AH, Panchal R, Mitchell DA, Moody PCE, Wallis R (2011) Structural basis of Mannan-binding lectin recognition by its associated serine protease MASP-1: implications for complement activation. Structure 19(11):1635–1643. https://doi.org/10.1016/j.str.2011.08.014

    Article  CAS  PubMed  Google Scholar 

  29. Kang JH, Super M, Yung CW, Cooper RM, Domansky K, Graveline AR, Mammoto T, Berthet JB, Tobin H, Cartwright MJ, Watters AL, Rottman M, Waterhouse A, Mammoto A, Gamini N, Rodas MJ, Kole A, Jiang A, Valentin TM, Diaz A, Takahashi K, Ingber DE (2014) NAT MED 20(10):1211-1216.https://doi.org/10.1038/nm.3640

  30. Xue YY, Shi HM, Feng B, Qiao L, Ding CAF, Yu SN (2021) Rapid identification of bacteria directly from blood cultures by Co-magnetic bead enrichment and MALDI-TOF MS profiling. Talanta 233:122472. https://doi.org/10.1016/j.talanta.2021.122472

    Article  CAS  PubMed  Google Scholar 

  31. Zhang Y, Yu M, Zhang C, Ma WF, Zhang YT, Wang CC, Lu HJ (2014) Highly selective and ultra fast solid-phase extraction of N-glycoproteome by oxime click chemistry using aminooxy-functionalized magnetic nanoparticles. Anal Chem 86(15):7920–7924. https://doi.org/10.1021/ac5018666

    Article  CAS  PubMed  Google Scholar 

  32. Moyes Rita B, Reynolds Jackie, Breakwell Donald P (2009) Differential staining of bacteria: Gram stain. Current protocols in microbiology Appendix: Appendix 3C.https://doi.org/10.1002/9780471729259.mca03cs15

  33. Bahr NC, Nuwagira E, Evans EE, Cresswell FV, Bystrom PV, Byamukama A, Bridge SC, Bangdiwala AS, Meya DB, Denkinger CM, Muzoora C, Boulware DR (2018) Diagnostic accuracy of Xpert MTB/RIF Ultra for tuberculous meningitis in HIV-infected adults: a prospective cohort study. Lancet Infect Dis 18(1):68–75. https://doi.org/10.1016/S1473-3099(17)30474-7

    Article  PubMed  PubMed Central  Google Scholar 

  34. Kuppermann N, Dayan PS, Levine DA, Vitale M, Tzimenatos L, Tunik MG, Saunders M, Ruddy RM, Roosevelt G, Rogers AJ, Powell EC, Nigrovic LE, Muenzer J, Linakis JG, Grisanti K, Jaffe DM, Hoyle JD, Greenberg R, Gattu R, Cruz AT, Crain EF, Cohen DM, Brayer A, Borgialli D, Bonsu B, Browne L, Blumberg S, Bennett JE, Atabaki SM, Anders J, Alpern ER, Miller B, Casper TC, Dean JM, Ramilo O, Mahajan P (2019) A clinical prediction rule to identify febrile infants 60 days and younger at low risk for serious bacterial infections. Jama Pediatr 173(4):342–351. https://doi.org/10.1001/jamapediatrics.2018.5501

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported by Moutai Group Research and Development Project (No. 2018023) and National Natural Science Foundation of China (31470786).

Author information

Authors and Affiliations

  1. Key Laboratory of Advanced Mass Spectrometry and Molecular Analysis of Zhejiang Province, Institute of Mass Spectrometry, School of Material Science and Chemical Engineering, Ningbo University, Ningbo, 315211, Zhejiang, China

    Menghuan Yu, Haimei Shi & Shaoning Yu

  2. Peking Union Medical College & Chinese Academy Medical Science, Department of Clinical Lab, Peking Union Medical College Hospital, Beijing, 100730, China

    Li Zhang

  3. Department of Infectious Diseases, Ningbo First Hospital, Ningbo University, Ningbo, 315211, Zhejiang, China

    Guoqing Qian, Haimei Shi & Shaoning Yu

Authors
  1. Menghuan Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Li Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guoqing Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Haimei Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shaoning Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Material preparation, data collection and analysis were performed by Menghuan Yu, Li Zhang, and Guoqing Qian. The first draft of the manuscript was written by Menghuan Yu and all authors commented on previous versions of the manuscript. Shaoning Yu and Haimei Shi contributed to the study conception and design and took primary responsibility for communication with the journal during the manuscript submission, peer-review, and publication process. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Haimei Shi or Shaoning Yu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 37807 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yu, M., Zhang, L., Qian, G. et al. Fc-MBL-modified Fe3O4 magnetic bead enrichment and fixation in Gram stain for rapid detection of low-concentration bacteria. Microchim Acta 189, 169 (2022). https://doi.org/10.1007/s00604-022-05277-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00604-022-05277-7

Keywords

Search

Navigation