Pharmaceutical Microbiology

ANTIMICROBIAL SUSCEPTIBILITY TESTING

Written by MicroDok

Antimicrobial susceptibility testing (AST) is a test that is used to determine the specific antibiotic/drug that can be used to inhibit or kill a microbial cell. It helps to guide a medical doctor on the best drug of choice and dosage to be prescribed for a given infection. Antibiogram is another name for antimicrobial susceptibility testing. AST is a very significant protocol in the clinical microbiology laboratory because it is the basis upon which drug prescription can be made for a patient suffering from a given ailment. It is worthy of note that antibiogram is carried out only on known and identified pathogens. It is wrong to carry out AST on non-pathogenic strains of microbes including commensals isolated from patients specimens. Though viruses, fungi, and protozoa can also be identified and characterized in the clinical microbiology laboratory, the apparatus with which to undertake AST on these pathogens are not routinely available in the laboratory as it is with bacterial pathogens that can be routinely identified and tested for antibiogram.

The main aim or reason of testing pathogenic microorganisms for their susceptibility to antibiotics is to be able to detect any form of drug resistance in the pathogens being tested, and also to select the appropriate drugs to which the pathogens will be susceptible to in the course of therapy. Due to increase in the morbidity and mortality rate of infectious diseases caused by drug resistant pathogens, AST should be a routine in every clinical m microbiology laboratory so as to keep the development and spread of drug resistant microbes at bay. The resistance of microbes to some empirical antimicrobial agents portends grave danger to the health sector and even to the economy of any nation due to increase in the cost of treatment and the possibility of treatment failures that may result from therapy.

Thus antibiogram should continue to be an integral part of the practice of clinical microbiology laboratories around the world, and it should always be performed on individual pure isolates of pathogenic microorganisms so that therapy can be properly guided. The introduction of several antibiotics into clinical medicine coupled with the increasing rate of the emergence of resistant pathogens necessitates the need to routinely carry out AST in the clinical microbiology laboratory in order to sustain the shelf-life of these drugs. Factors that can affect the result of antimicrobial susceptibility testing include: inoculum size of the test organism, pH, temperature, moisture and effects of thymidine or thymine.

The results of antibiogram are usually reported as minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC), and each of these results are further categorized as susceptible, intermediate or resistant depending on the inhibition zone diameter (IZD) produced by the tested drug on the test pathogen. MIC is the lowest concentration of a drug/antibiotic that can inhibit the growth of a microorganism while MBC is the lowest concentration of an antibiotic that can kill a microorganism.. According to the Clinical and Laboratory Standard Institute (CLSI), AST is carried out in the laboratory by any of the following methods:

1. Disk diffusion method: Disk diffusion method is a simple and practical AST that is widely carried out in the clinical microbiology laboratory. It requires no special equipment to carry out, and it is one of the oldest means of testing a wide range of bacterial pathogens for susceptibility to drugs. The Kirby-Bauer method is a typical example of disk diffusion method. Disk diffusion method is carried out by applying a bacterial inoculum (adjusted to 0.5 McFarland turbidity standards) on a Mueller-Hinton or nutrient agar plate. The plate is allowed for some minutes, after which commercially available paper disk(s) containing known concentration of antibiotics is aseptically applied to the plate. The plate is incubated, and the IZD around each of the disk is measured to the nearest millimeter using a caliper or meter rule. Basically the IZDs obtained are compared to known breakpoints of the tested drugs as per the CLSI criteria in order to know if the organism is susceptible, intermediate or resistant to the drugs being tested.

Illustration of susceptibility test plate of a Pseudomonas aeruginosa isolate, showing different levels of susceptibility and resistance of the test organism to the test antibiotics (as detected by disk diffusion method)

2. Agar dilution and diffusion method: The agar dilution and diffusion susceptibility test is usually performed using the epsilometer (E) test strip. E-test is an exponential gradient susceptibility testing procedure that combines both dilution and diffusion techniques in the determination of antibiogram. It is a quantitative testing method that makes use of an inert test strip that is incorporated with predefined antimicrobial agents that produces a symmetrical inhibition ellipse after incubation. E-test can be used to determine the antibiogram of both fastidious and non-fastidious bacterial pathogens.

3. Dilution susceptibility method: Dilution susceptibility test is used to determine the minimal amount of an antimicrobial agent that will be required to either kill or inhibit the growth of a bacterial pathogen. They are mainly used to determine the MICs and MBCs of empirical drugs/antibiotics on specific bacterial pathogens unlike the disk diffusion method that determine the IZD of a drug. Examples of dilution tests include: broth dilution, agar dilution, and macro-broth dilution methods. The antimicrobial agents are usually diluted in either broth or agar and tested at a two-fold serial dilution in order to determine their susceptibility pattern in relation to specific bacterial pathogens. Today, there exist several automated and rapid methods of conducting antibiogram tests in the clinical microbiology laboratory. Some of these methods include epsilometer (E)-test, Vitek MIC test, the Sensititre ARIS 2X Automated Systems, BD Phoenix Automated Microbiology Systems, and so on.

McFarland Turbidity Standards

McFarland Turbidity Standards are series of standards which are used as references to adjust the turbidity of bacterial suspensions during antimicrobial susceptibility testings so that the actual amount of inoculum size used will be within a certain limit and be known at the same time. They are used as reference standards in the preparations of suspensions of bacterial cultures. McFarland turbidity standards are used to know the actual number of bacteria that is present in a liquid culture or suspension by comparing the suspension to a known standard. Basically, McFarland turbidity standards are composed or made up of three main ingredients which are barium chloride and concentrated tetraoxosulphate (vi) acid. McFarland turbidity standards is one of the easiest method of estimating or determining the actual amount of the test bacteria required to undertake an antimicrobial susceptibility testing.

PROTOCOL FOR MINIMUM INHIBITORY CONCENTRATION (MIC): 2-FOLD SERIAL DILUTION

Minimum inhibitory concentration (MIC) is defined as the minimum amount or concentration of antibiotics and/or drug that is required to kill or inhibit the growth of a pathogen either in vivo or in vitro. The procedure involved in determining the MIC of antimicrobial agents against a test organism in vitro is succinctly enumerated as follows:

  1. Set up 5 test tubes or more in a test tube rack and label them tube A, tube B, tube C, tube D and tube E as the case may be.
  2. Set up positive and negative control tubes. Negative control tube contains the bacterial culture and the Mueller-Hinton (MH) broth and remains turbid. Positive control tube is clear and contains only the MH broth.
  3. Add 0.5 ml of Mueller-Hinton (MH) broth to each of the tubes (A-E). This should be done with a sterile pipette.
  4. Add 0.5 ml of the test antibiotic (from a stock concentration of 250 mg/ml) to tube A. Mix the solution properly. Upon dilution tubes A, B, C, D & E will be 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, 3.125 mg/ml and 1.563 mg/ml.
  5. From tube A, transfer 0.5 ml of the diluted antibiotic solution to tube B.
  6. Repeat this process until all the tubes have been covered.
  7. Dispense or discard 0.5 ml of the diluted antibiotic solution from tube E.
  8. Transfer 0.5 ml of bacterial suspension (adjusted to 0.5 McFarland turbidity standards) to each of the tubes (labeled A-E). Mix the content of each tube properly.
  9. Incubate the tubes in the test tube rack at 37oC overnight.
  10. After incubation, check each of the tubes for turbidity or cloudiness. Ensure not to shake the tubes when doing so.
  11. Look out for the tube without turbidity or cloudiness. This tube without cloudiness or turbidity is the MIC of the antibiotic for the test bacteria. Absence of cloudiness is indicative of the inhibitory effect of the test agent against the bacteria in broth culture.

PROTOCOL FOR DISK DIFFUSION TEST

The disk diffusion test provides a qualitative evaluation of bacterial growth inhibition by a given antibiotic. In the disk diffusion test, the antibiotic concentrations are created by diffusion of the drug through a paper disk containing known concentration of the drug. The zone of inhibition produced after incubation of the agar plate is used to determine the susceptibility or resistance profile of the test bacteria to the antibiotic qualitatively. Disk diffusion technique involves the following simplified steps:  

  1. Inoculate the test bacteria (adjusted to 0.5 McFarland turbidity standards) on agar plate. This process is carried out by swabbing the suspension of the test bacteria on the entire surface of the agar plate.
  2. Aseptically place the disk on the swabbed agar plate.
  3. Allow plates for about 10-20 min for pre-diffusion of the drug to occur. Antibiotic disks containing defined concentration of drugs are commercially available. But susceptibility disks can be prepared in the laboratory if the researcher so chooses.
  4. Incubate the plate at 37oC for 24 hrs or overnight as the case may be.
  5. After incubation, check the plates for zones of inhibition.
  6. Measure the inhibition zone diameter (IZD) using a meter rule. The unit of the IZD should be in millimeter (mm).
  7. Record the readings or values obtained according to the CLSI criteria, and report the bacteria as susceptible, intermediate or resistant to the test antibiotic(s).

REFERENCES

World Health Organization (WHO). Strategy for Containment of Antimicrobial Resistance. WHO/CDS/CR/DRS/2001.2.

Washington J.A (1993). Rapid antimicrobial susceptibility testing: technical and clinical considerations. Clin Microbiol Newsl, 15:153–155.

Walsh T.R., Toleman M.A., Poirel L and Nordmann P (2005). Metallo β – Lactamases: the Quiet before the Storm? Clinical Microbiology Review, 18(2):306-325.

Thomson K.S and Moland E.S (2000). The new b-lactamases of Gram-negative bacteria at the dawn of the new millennium (Review). Microbes and Infection, 2:1225-1235.

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

Pitout J.D.D and Laupland K.B (2008). Extended – spectrum β – lactamase – producing Enterobacteriaceae:  an emerging public health concern. Lancet Infect Dis, 8:159-66.

Livermore D.M (2004). The need for new antibiotics. Clinical Microbiology & Infection, 4(10): 1-9.

Levy S.B and Marshall B (2004). Antibacterial resistance worldwide: Causes, challenges and responses. Nature Medicine, 12(10):S122-S129.

Jayaramah R (2009). Antibiotic Resistance: an overview of mechanisms and a paradigm shift. Current Science, 96(11):1475-1484.

Jacoby G.A and Munoz-Price L.S (2005). Mechanisms of Disease: The New β – lactamases. N Engl J Med, 352:380-91.

Hart C.A (1998). Antibiotic Resistance: an increasing problem? BMJ, 316:1255-1256.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

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.

Denyer S.P., Hodges N.A and Gorman S.P (2004). Pharmaceutical Microbiology. 7th ed. Blackwell Publishing Company, USA.

About the author

MicroDok

Leave a Comment