top of page
REVIEW ARTICLE

      Abstract    

        

HOME

BIOCHEMICAL AND MOLECULAR APPROACHES TO BACTERIAL PATHOGEN IDENTIFICATION: TRADITIONAL TECHNIQUES AND EMERGING ADVANCES

​

Subhajit Karmakar, Athar Rehan, Rukshar Jahan, Mahenoor

​​

ABSTRACT:

In clinical microbiology, monitoring the environment and protecting food safety, having the ability to identify bacteria is critical for learning about infectious diseases, the growth of microbes and pathogens found in food. Tests that rely on bacteria’s metabolic processes are used for telling apart and recognizing different kinds of bacteria. I will explain traditional and modern biochemical tests, what they involve, their strengths and their weaknesses. It looks at how microbiology is applied to clinical diagnostics, environmental monitoring and the food and drinks industry, handling problems related to using phenotypic methods and explaining recent developments that improve both. The review points out that reliability, reproducibility and similarity between test results from different laboratories depend on strict quality control and set protocols. Thanks to this, ribosomal proteins are still important in diagnosing with molecular methods and add to our understanding of bacteria and their social interactions

Keywords: Clinical microbiology, Infectious diseases, Metabolic processes, Biochemical tests, Clinical diagnostics, Phenotypic methods, Quality control, Molecular methods.

​

​

REFERENCES:​​

  1. Li H, Torab P, Mach KE, Surrette C, England MR, Craft DW, Thomas NJ, Liao JC, Puleo C, Wong PK. Adaptable microfluidic system for single-cell pathogen classification and antimicrobial susceptibility testing. Proceedings of the National Academy of Sciences. 2019 May 21;116(21):10270-9.

  2. Faron ML, Buchan BW, Hyke J, Madisen N, Lillie JL, Granato PA, Wilson DA, Procop GW, Novak-Weekley S, Marlowe E, Cumpio J. Multicenter evaluation of the Bruker MALDI Biotyper CA system for the identification of clinical aerobic gram-negative bacterial isolates. PLoS One. 2015 Nov 3;10(11):e0141350.

  3. Bhattacharyya RP, Walker M, Boykin R, Son SS, Liu J, Hachey AC, Ma P, Wu L, Choi K, Cummins KC, Benson M. Rapid identification and phylogenetic classification of diverse bacterial pathogens in a multiplexed hybridization assay targeting ribosomal RNA. Scientific reports. 2019 Mar 14;9(1):4516.

  4. Endimiani A, Ramette A, Rhoads DD, Jacobs MR. The evolving role of the clinical microbiology laboratory in identifying resistance in Gram-negative bacteria: an update. Infectious disease clinics of North America. 2020 Dec 1;34(4):659-76.

  5. Semret M, Ndao M, Jacobs J, Yansouni CP. Point-of-care and point-of-‘can’: leveraging reference-laboratory capacity for integrated diagnosis of fever syndromes in the tropics. Clinical Microbiology and Infection. 2018 Aug 1;24(8):836-44.

  6. Roca I, Akova M, Baquero F, Carlet J, Cavaleri M, Coenen S, Cohen J, Findlay D, Gyssens I, Heure OE, Kahlmeter G. The global threat of antimicrobial resistance: science for intervention. New microbes and new infections. 2015 Jul 1;6:22-9.

  7. Sanchini A. Recent developments in phenotypic and molecular diagnostic methods for antimicrobial resistance detection in Staphylococcus aureus: a narrative review. Diagnostics. 2022 Jan 15;12(1):208.

  8. Franco-Duarte R, Černáková L, Kadam S, S. Kaushik K, Salehi B, Bevilacqua A, Corbo MR, Antolak H, Dybka-Stępień K, Leszczewicz M, Relison Tintino S. Advances in chemical and biological methods to identify microorganisms—from past to present. Microorganisms. 2019 May 13;7(5):130.

  9. Elston HR, Baudo JA, Stanek JP, Schaab M. Multi-biochemical test system for distinguishing enteric and other gram-negative bacilli. Applied microbiology. 1971 Sep;22(3):408-14.

  10. MacWilliams MP. Citrate test protocol. American Society for Microbiology. 2009 Dec 8:1-7.

  11. Rahman MA, Ahmad T, Mahmud S, Barman NC, Haque MS, Uddin ME, Ahmed R. Isolation, identification and antibiotic sensitivity pattern of Salmonella spp. from locally isolated egg samples. Am. J. Pure Appl. Sci. 2019;1(1):1-1.

  12. Taylor WI, Achanzar D. Catalase test as an aid to the identification of Enterobacteriaceae. Applied microbiology. 1972 Jul;24(1):58-61.

  13. Heston TF. A case study in blockchain health care innovation. International Journal of Current Research. 2017;9(11):60587-8.

  14. Al-Kharousi ZS, Al-Ramadhani Z, Al-Malki FA, Al-Habsi N. Date Vinegar: First Isolation of Acetobacter and Formulation of a Starter Culture. Foods. 2024 Apr 30;13(9):1389.

  15. Barer MR, Marsh PJ. Rapid cytochemical demonstration of cytochrome oxidase activity in pathogenic bacteria. Journal of clinical pathology. 1992 Jun 1;45(6):487-9.

  16. JURTSHUK JR PE, McQUITTY DN. Survey of oxidase-positive and-negative bacteria using a quantitative Kovacs oxidase test. International Journal of Systematic and Evolutionary Microbiology. 1976 Apr;26(2):127-37.

  17. Giuliano C, Patel CR, Kale-Pradhan PB. A guide to bacterial culture identification and results interpretation. Pharmacy and Therapeutics. 2019 Apr;44(4):192.

  18. Isenberg HD. Clinical microbiology procedures handbook. American Society of Microbiology; 1992.

  19. Rasouli Z, Abdollahi H, Maeder M. Generalized indicator-based determination of solution pH. Analytica Chimica Acta. 2020 May 1;1109:90-7.

  20. Cox NA, Thomson JE, Bailey JS. Minitek inoculum broth for testing indole production by Enterobacteriaceae. Journal of Food Protection. 1981 Jun 1;44(6):445-6.

  21. Di C, Yu W, Lu Y. Screening of Methanotrophic Strain for Scale Applications: Methane Emission Reduction and Resource Utilization. Sustainability. 2025 Apr 18;17(8):3687.

  22. Blount ZD, Maddamsetti R, Grant NA, Ahmed ST, Jagdish T, Baxter JA, Sommerfeld BA, Tillman A, Moore J, Slonczewski JL, Barrick JE. Genomic and phenotypic evolution of Escherichia coli in a novel citrate-only resource environment. Elife. 2020 May 29;9:e55414.

  23. Padron AP, Dockstader WB. Selective medium for hydrogen sulfide production by salmonellae. Applied Microbiology. 1972 Jun;23(6):1107-12.

  24. Küster E, Williams ST. Production of hydrogen sulfide by streptomycetes and methods for its detection. Applied microbiology. 1964 Jan;12(1):46-52.

  25. McDonough PL, Shin SJ, Lein DH. Diagnostic and public health dilemma of lactose-fermenting Salmonella enterica serotype Typhimurium in cattle in the Northeastern United States. Journal of clinical microbiology. 2000 Mar 1;38(3):1221-6.

  26. from a Patient SN. Isolation of Salmonella enterica Serovar. (2014)

  27. Franklin ML, Clark WA. Simple, inexpensive, and rapid way to produce Bacillus subtilis spores for the Guthrie bioassay. Journal of Clinical Microbiology. 1981 Jul;14(1):113-5.

  28. Matsuo H, Hanamure Y, Miyano R, Takahashi Y, Ōmura S, Nakashima T. Screening for sulfur compounds by molybdenum-catalyzed oxidation combined with liquid chromatography-mass spectrometry. Molecules. 2020 Jan 7;25(2):240.

  29. Starr SE, Thompson FS, Dowell Jr VR, Balows A. Micromethod system for identification of anaerobic bacteria. Applied Microbiology. 1973 May;25(5):713-7.

  30. Zengin Canalp H, Bayraktar B. Direct rapid identification from positive blood cultures by MALDI-TOF MS: Specific focus on turnaround times. Microbiology Spectrum. 2021 Dec 22;9(3):e01103-21.

  31. Ping Y, Chen Q, Sun X, Wang H, Lin S, Zhu B, Wang Z, Lu J, Cao J. Clinical evaluation of an in-house Xpert Lysate-based Method combined with MALDI-TOF MS for the rapid identification of positive blood cultures. BMC microbiology. 2025 Dec;25(1):1-2.

  32. Clark CM, Murphy BT, Sanchez LM. A call to action: the need for standardization in developing open-source mass spectrometry-based methods for microbial subspecies discrimination. Msystems. 2020 Feb 25;5(1):10-128.

  33. Kohlmann R, Hoffmann A, Geis G, Gatermann S. MALDI-TOF mass spectrometry following short incubation on a solid medium is a valuable tool for rapid pathogen identification from positive blood cultures. International Journal of Medical Microbiology. 2015 Jun 1;305(4-5):469-79.

  34. Procop GW. Molecular diagnostics for the detection and characterization of microbial pathogens. Clinical Infectious Diseases. 2007 Sep 1;45(Supplement_2):S99-111.

  35. Senok A, Dabal LA, Alfaresi M, Habous M, Celiloglu H, Bashiri S, Almaazmi N, Ahmed H, Mohmed AA, Bahaaldin O, Elimam MA. Clinical impact of the BIOFIRE blood culture identification 2 panel in adult patients with bloodstream infection: a multicentre observational study in the United Arab Emirates. Diagnostics. 2023 Jul 21;13(14):2433.

  36. Maeda Y, Sugiyama Y, Kogiso A, Lim TK, Harada M, Yoshino T, Matsunaga T, Tanaka T. Colony fingerprint-based discrimination of Staphylococcus species with machine learning approaches. Sensors. 2018 Aug 24;18(9):2789.

  37. Xu F, Chen C, Lu S, Xue M, Ding H, Song Y, Zhang Y, Sun K, Tang L, Wang W, Wang M. Impact of metagenomics next-generation sequencing on etiological diagnosis and early outcomes in sepsis. Journal of Translational Medicine. 2025 Apr 3;23(1):394.

  38. Al-Zahrani IA. Routine detection of carbapenem-resistant gram-negative bacilli in clinical laboratories: A review of current challenges. Saudi medical journal. 2018 Sep;39(9):861.

  39. Jacobs MR, Colson JD, Rhoads DD. Recent advances in rapid antimicrobial susceptibility testing systems. Expert Review of Molecular Diagnostics. 2021 Jun 3;21(6):563-78.

  40. Piddock LJ. Assess drug-resistance phenotypes, not just genotypes. Nature microbiology. 2016 Jul 26;1(8):1-2.

  41. Ceuppens S, Delbeke S, De Coninck D, Boussemaere J, Boon N, Uyttendaele M. Characterization of the bacterial community naturally present on commercially grown basil leaves: evaluation of sample preparation prior to culture-independent techniques. International Journal of Environmental Research and Public Health. 2015 Aug;12(8):10171-97.

  42. Forbes BA, Sahm DF, Weissfeld AS, Bailey SS. Diagnostic microbiology 12th Edition: Mosby Elsevier, St. Louis, MO. 2007:778-81.

  43. Cheesbrough M. Salmonellae. District Laboratory Practice in Tropical Countries (Part 2), 2nd Edition Updated. Cambridge University Press,. Cape Town, South Africa. 2006:167.

  44. Ombelet S, Ronat JB, Walsh T, Yansouni CP, Cox J, Vlieghe E, Martiny D, Semret M, Vandenberg O, Jacobs J, Lunguya O. Clinical bacteriology in low-resource settings: today's solutions. The Lancet Infectious Diseases. 2018 Aug 1;18(8):e248-58.

  45. Peeling RW, Mabey D. Point-of-care tests for diagnosing infections in the developing world. Clinical microbiology and infection. 2010 Aug 1;16(8):1062-9.

  46. Yager P, Domingo GJ, Gerdes J. Point-of-care diagnostics for global health. Annu. Rev. Biomed. Eng.. 2008 Aug 15;10(1):107-44.

  47. Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, Allen J, Tahirli R, Blakemore R, Rustomjee R, Milovic A. Rapid molecular detection of tuberculosis and rifampin resistance. New England Journal of Medicine. 2010 Sep 9;363(11):1005-15.

  48. Niemz A, Ferguson TM, Boyle DS. Point-of-care nucleic acid testing for infectious diseases. Trends in biotechnology. 2011 May 1;29(5):240-50.

  49. World Health Organization, Global Diagnostics Landscape and Priorities, 2021.

  50. Damor RR, Kubavat AR. Antibiotic sensitivity profile of Klebsiella isolates and it’s impact on clinical outcome

 To cite this article:

Karmakar S, Rehan A, Jahan R, Mahanoor. Biochemical and molecular approaches to bacterial pathogen identification: traditional techniques and emerging advances. Int. J. Med. Lab. Res. 2025;10,2:34-42. http://doi.org/10.35503/IJMLR.2025.10205

bottom of page