PHENOTYPIC AND GENOTYPIC DETECTION METHODS FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING (AST)

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It is noteworthy that the performance of antimicrobial susceptibility testing on putative clinical bacterial or fungal pathogens is vital to confirm the susceptibility of the individual organisms to some chosen empirical antibiotics. More so, antimicrobial susceptibility testing will help the physician and/or laboratory scientist to detect resistance in the individual test organism so that antimicrobial therapy can be properly guided. The expression “phenotypic” is from the word phenotype, which means “the observable characteristics of an organism”. It describes the physical features of an organism in terms of its morphology, motility, colour and shape amongst other noticeable attributes that are typical to a particular organism. The phenotypic detection and characterization of antimicrobial resistant genes in pathogenic bacteria describes the general physical reaction or observable traits of microbes to a variety of antimicrobial agents such as antibiotics, disinfectants and antiseptics.

In phenotypic detection, pathogenic strains of microorganisms are directly tested in vitro for their susceptibility or resistance to a range of antimicrobial agents (especially antibiotics) that are usually used for their control. The result obtained from a phenotypic detection and characterization experiment generally gives a picture of the performance of the test drugs in vivo (i.e. when used for the practical treatment of a sick host). Phenotypic detection methods provides prompt and reliable drug susceptibility results that give the microbiologist a picture of the prevailing resistant profile of the test pathogen. This guides physicians on the choice of antimicrobial therapy to initiate in the patient. Phenotypic detection and characterization techniques used in the microbiology laboratory to classify resistant genes of pathogenic bacteria are easy to perform and report than their genotypic counterparts.

Some phenotypic detection methods (e.g. the VITEK system) are automated and can be used to perform computerized or automated antimicrobial susceptibility testing in the microbiology laboratory. The MicroScan system, VITEK 1 and 2, Sensititre ARIS 2X and the BD Phoenix Automated Microbiology Systems are typical examples of available automated antimicrobial susceptibility methods that can be used in the microbiology laboratory to promptly evaluate the susceptibility profile of pathogenic bacteria based on their MIC values. These automated antimicrobial susceptibility testing systems have automated computer software used for the interpretation of susceptibility test results. They also have “expert systems” which can be used to analyze test results for atypical or unusual susceptibility patterns as well as for uncommon resistance phenotypes amongst the test microbes.

Rapid antimicrobial susceptibility testing results are critical for total patient outcome. This is because such results when timely obtained, can lead to choosing the right antimicrobial therapy for each patient. It helps to prevent blind treatment – which may allow resistant microbial strains to emerge and spread. Phenotypic detection and characterization techniques for the identification of resistance genes in pathogenic bacteria are generally less expensive and straightforward to perform. Most importantly, they can be used to monitor the progress and response of a diseased patient to antimicrobial therapy. One of the extensively used and generally accepted methods of characterizing the resistance phenotypes of pathogenic bacteria by phenotypic methods in the microbiology laboratory is the Kirby-Bauer disk diffusion technique. The broth dilution test and antimicrobial gradient methods are other non-automated techniques of determining the susceptibility patterns of microbes to antimicrobial agents inclusive of the disk diffusion technique as aforesaid.

Genotypic antimicrobial susceptibility testing (AST) techniques uses molecular biology tools/techniques such as PCR and sequencing amongst others to identify specific resistance genes or genetic mutations that are responsible for the resistance of pathogens to antimicrobial agents or antibiotics. Generally, the genotypic techniques of AST are very fast unlike the phenotypic techniques that are usually slower in detecting resistance phenotypes. While the phenotypic techniques of AST are culture-based, the genotypic AST techniques are not culture-based, but uses molecular biology techniques to identify the genes or genetic factors responsible for the resistance in the microorganism being investigated.

DNA-based, amplification-based, or sequencing-based methods are employed in the genotypic techniques of AST. The goal of the genotypic methods of AST is to unravel the genetic mechanisms or genes responsible for the resistance of the test isolates to the antimicrobial agent or antibiotic. Genotypic AST can be used to validate the results of phenotypic AST in the microbiology laboratory.

Examples of the genotypic AST techniques include:

  • Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) 
  • Hybridization-Based Systems such as FISH (fluorescent in situ hybridization)
  • Nucleic Acid Amplification Technology (NAAT) such as quantitative PCR (qPCR)
  • Immunodetection of resistant pathogens
  • Whole Genome Sequencing techniques
  • LAMP (loop-mediated isothermal amplification) techniques
  • Use of DNA microarray techniques

FURTHER READING

Ashutosh Kar (2008). Pharmaceutical Microbiology, 1st edition. New Age International Publishers: New Delhi, India. 

Block S.S (2001). Disinfection, sterilization and preservation. 5th edition. Lippincott Williams & Wilkins, Philadelphia and London.

Courvalin P, Leclercq R and Rice L.B (2010). Antibiogram. ESKA Publishing, ASM Press, Canada.

Denyer S.P., Hodges N.A and Gorman S.P (2004). Hugo & Russell’s Pharmaceutical Microbiology. 7th ed. Blackwell Publishing Company, USA. Pp.152-172.

Ejikeugwu Chika, Iroha Ifeanyichukwu, Adikwu Michael and Esimone Charles (2013). Susceptibility and Detection of Extended Spectrum β-Lactamase Enzymes from Otitis Media Pathogens. American Journal of Infectious Diseases. 9(1):24-29.

Finch R.G, Greenwood D, Norrby R and Whitley R (2002). Antibiotic and chemotherapy, 8th edition. Churchill Livingstone, London and Edinburg.

Russell A.D and Chopra I (1996). Understanding antibacterial action and resistance. 2nd edition. Ellis Horwood Publishers, New York, USA.

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