Antibiotic resistant is slowly reaching for the top as a public health threat. It is therefore important to keep educating and enlightening the public about this menace and solutions to defeat it

1. EFFECTIVE ANTIMICROBIAL SUSCEPTIBILITY
TESTING; A PATH TO SOLVING THE ANTIMICROBIAL RESISTANCE
MENACE GLOBALLY
A SEMINAR PRESENTATION AT DEPARTMENT OF MEDICAL LABORATORY SERVICES
HOSPITAL MANAGEMENT BOARD
BY
ADEKILEKUN HAMIDAH HAROON
SUPERVISED BY MR OKOLI CHUKWUDINMA (ADMLS)
GOOD LABORATORY DESK OFFICER
OCTOBER, 2022

2. Outline
2
⬢ Aim of presentation
⬢ Overview of Antimicrobials
⬢ Antimicrobial resistance (AMR)
⬢ Mechanism of AMR used by bacteria
⬢ Causes of AMR
⬢ Antimicrobial Susceptibility Testing
⬢ Standard Laboratory Procedure for AST
⬢ Challenges and Solutions
⬢ Conclusion
⬢ Recommendation
⬢ References

3. Aim of Presentation
3
The aim is to educate us on how effective Antimicrobial Susceptibility
Testing (AST) can serve as a path towards solving Antimicrobial
Resistance (AMR) globally.

4. Antimicrobials: Overview
4
⬢ Antimicrobial agents are medicines that stops or inhibits the growth of microorganisms
⬢ They include antibiotics, antivirals, antifungals and antiparasitic depending on the type of
microorganisms they work against
⬢ Dating back to the ancient Egyptians, humans have been using natural plants, herbs and even
mold to cure different ailments
⬢ However, the first antimicrobial agent to be used was pyocyanase, a by-product of bacillus
pyocyaneus in 1888 by a German scientist, E de freudenreich which was later discontinued
⬢ Followed by the discovery of Salvarsan in 1910 by Paul Ehrlich, the first substance to
successfully cure an infection, syphilis!
⬢ Followed by the discovery of Penicillin in 1928 by Fleming which started the golden age of natural
product antibiotic
⬢ Antimicrobials have existed for up to 100 years now and have been regarded as one of the
wonders of modern medicine which is said to have extended the average human lifespan by 23
years
1

5. Antibiotics
⬢ Antibiotics, also known as antibacterial agents are a type of antimicrobial
that kill or inhibit the growth of bacteria
⬢ They are divided into two major groups; broad-spectrum antibiotics and narrow-spectrum
antibiotics
⬢ Broad-spectrum antibiotics act on a wide range of disease-causing bacteria e.g.
aminoglycosides, cephalosporins, quinolones etc.
⬢ Narrow-spectrum antibiotics are active against a selected group of bacterial types e.g.
vancomycin , macrolides
⬢ They can also be grouped by their mode of action i.e. their ability to inhibit the synthesis of
the cell wall, cell membrane, proteins, and nucleic acids of bacteria
⬢ Examples of popular antibiotics include: amoxicillin, doxycycline, ciprofloxacin,
metronidazole, sulfamethoxazole and trimethoprim and many more
1
5

6. Classes And Families of Antibiotics
Penicillins:
Inhibitors of bacterial cell
wall synthesis
⬢ Natural penicillins (penicillin
G, penicillin V)
⬢ B-lactamase resistant
penicillins - antistaphylococcal
(methicillin, cloxacillin)
⬢ Aminopenicillins
(Amoxicillin, Ampicillin)
⬢ Extented-spectrum
penicillins – antipseudomonal
(Carbenicillin, Pipercillin,
Ticarcillin)
Fluoroquinolones:
Inhibitors of nucleic acid
synthesis
⬢ 1st Gen (Nalidixic acid,
Cinoxacin)
⬢ 2nd Gen (Norfloxacin,
Ciprofloxacin, Enoxacin,
Ofloxacin)
⬢ 3rd Gen (Levofloxacin,
Sparfloxacin, Moxifloxacin,
Gemifloxacin)
⬢ 4th Gen (Trovafloxacin)
6
Cephalosporins: Inhibitors
of bacterial cell wall
synthesis
⬢ 1st Gen (Cephalexin,
Cefazolin etc.)
⬢ 2nd Gen (Cefoxitin,
Cefuroxime etc.)
⬢ 3rd Gen (Cefotaxime,
Ceftriaxone etc.)
⬢ 4th Gen (Cefepime,
Cefquinone etc.)
⬢ 5th Gen (Ceftaroline etc.)
Lincosamides:
Inhibitors of protein
synthesis
⬢ Clindamycin
Glycopeptides:
Inhibitors of bacterial
cell wall synthesis
⬢ Vancomycin
⬢ Teicoplanin
Etc.

7. Carbapenems:
Inhibitors of bacterial
cell wall synthesis
⬢ Imipenem-cilastatin
⬢ Meropenem
⬢ Ertapenem
⬢ Doripenem
Etc.
Sulphonamides and
Trimethoprim:
Inhibitors of bacterial
nucleic acid synthesis
⬢ Cotrimoxazole
⬢ Trimethoprim
Etc.
7
Macrolides:
Inhibitors of protein
synthesis
⬢ Azithromycin
⬢ Clarithromycin
⬢ Erythromycin
Etc
Rifamycins:
Antimycobacterial
agents
⬢ Rifabutin
⬢ Rifampicin
⬢ Rifapentine
⬢ rifaximin
Oxazolidinones:
Inhibitors of protein
synthesis
⬢ Linezolid
⬢ Tedizolid
Aminoglycosides:
Inhibitors of protein
synthesis
⬢Gentamycin
⬢Amikacin
⬢Streptomycin
⬢Neomycin
⬢Kanamycin
Etc.

8. Look closely…
8
2009 2019

9. Antimicrobial Resistance
9
⬢ Antimicrobial resistance (AMR) occurs when bacteria, viruses, fungi and parasites change over
time and no longer respond to medicines they were previously responding to, making infections
harder to treat and increasing the risk of disease spread, raising cost of treatments as well and
finally to severe illnesses and death
⬢ AMR is said to be a significant public health and development threat which requires urgent
multisectoral action in order to achieve the Sustainable Development Goals (SDGs) (WHO, 2021)
⬢ In 2019, WHO declared AMR as one of the top 10 global public health threats facing humanity
⬢ Antibiotic resistance is a major subset of AMR, that applies specifically to bacteria that become
resistant to antibiotic
⬢ Antibiotic resistance could kill humanity before climate change does, warns World Health
Organization.
⬢ A growing list of infections such as – Pneumonia, Tuberculosis, blood poisoning, gonorrhea, and
foodborne diseases – are becoming harder, and sometimes impossible, to treat as antibiotics
become less effective

10. 10 million deaths
Annually by 2050
2-3 million
Infections per year
1.27 million deaths
In 2019
10
In 2019

11. Mechanism of AMR Used by Bacteria
11
Antimicrobial resistance mechanisms fall into four main categories:
⬢ Limiting intake of drug
⬢ Modifying drug target
⬢ Inactivating a drug
⬢ Active drug efflux

12. 12
CAUSES
OF AMR
Lack of
awareness
Limitation of
recent AMR
data
Inadequate
diagnostic
capacity
Improper
management of
waste and
sanitation
Insufficient
financial
resources
Absence of
proper
surveillance
systems
Misuse and
overuse of
antibiotics
Inadequate
infection control
practices in
hospital

13. Medical Laboratory Science Practice & Limitations
⬢ Inadequate organism identification
⬢ Isolation of normal bacterial flora as pathogenic
⬢ Substandard and/or inappropriate AST
⬢ Failure to carry on further AST in the case of MDR bacterium
⬢ Unskilled personnel as in charge
⬢ Sympathy susceptibility testing
⬢ Method of laboratory practices (not following all the CLSI standards)
13

14. 14
⬢ After isolation and identification of disease-causing bacteria during an MCS
test, what follows includes the test to detect the antibiotic the said bacteria is
sensitive or resistant to
⬢ Antimicrobial Susceptibility Testing (AST) is the procedure used to
determine which antimicrobials will inhibit the growth of the microorganism
causing a specific infection
⬢ AST is very important in confirming susceptibility, detecting resistance,
guiding selection for patient therapy and providing data for drug resistance
surveillance
⬢ The disc diffusion method based on Kirby-Bauer method is the simplest and
the most widely used method for AST and it has been shown to provide
reproducible and repeatable result when followed correctly
⬢ When this method is performed with strict adherence to standard
procedures in accordance to CLSI guidelines, it gives reliable results and
can predict clinical efficacy of the antibiotics
Antimicrobial Susceptibility Testing (AST)

15. Methods of AST
15
ANTIBIOTIC
SENSITIVITY
TESTS
DIFFUSION &
DILUTION
DIFFUSION
KIRBY-BAUER
METHOD
STOKES
METHOD
BROTH
DILUTION
AGAR
DILUTION
DILUTION
E-TEST
AUTOMATED
SYSTEMS
EG. VITEK
SYSTEMS

16. 16
Effective AST can be affected by multiple factors which include:
 The media
o the standard is Mueller Hinton Agar according to CLSI
o Thickness 4mm
o PH 7.2 – 7.4
 Antimicrobial discs or preparation
o Commercial disc or paper disc prepared according to the standard
o 100mm plate – 5 discs
o 150mm plate – Up to 12 discs
 Inoculum’s size
o 0.5% McFarland standard for inoculum preparation
o Prepared by 0.5ml 0.04mol/L BaCl2 and 99.5ml of 0.18mol/L of
H2SO4
 Incubation conditions
o Incubated at 370c for 16-18hrs
 Competence of the Medical laboratory scientist personnel
o Well trained personnel on AST
Effective Antimicrobial
Susceptibility Testing

17. 17
⬢ Prepare a standardized bacterial suspension from well-isolated
colonies using McFarland standards
⬢ Inoculate bacteria suspension on Mueller Hinton Agar (MHA) using a
sterile swab
⬢ Place antimicrobial discs on inoculated MHA depending on the
organisms isolated, not more than five discs on a 100mm plate or
twelve on a 150mm plate
⬢ Ensure discs are placed 15 mm from the edge of the plate and 24mm
between each other
⬢ Incubate plates at 370c for 16-18 hrs
⬢ Measure the zone of inhibition following the CLSI guidelines
⬢ Interprete AST using CLSI standards
Standard Laboratory Procedure for AST

18. Challenges and
Solutions
Challenges
 Cost
 Man power
 Technical know-how
 Laziness
 Inadequate and or substandard AST materials
Solutions
 Financing
 Adequate staffing
 Trainings, capacity building, workshops and
seminar
 Workplace discipline
 Standardization of locally produced AST materials
i.e. antibiotics 18
Some interesting
pictures…
CLSI D-TEST
POSITIVE PLATE
ESBL
POSITIVE PLATE
SUPER BUGS

19. Conclusion
Effective Antimicrobial Susceptibility testing
from the end of the medical laboratory
scientist will encourage events such as;
proper prescription of antibiotics to treat
patients’ infection, contribute to the data
available for control and prevention of
infectious diseases and also create a
means to monitor resistance patterns
caused by mutations in the bacterial DNA.
Antimicrobial Susceptibility Testing
performed according to standards is
therefore very important when considering
one of the pathways to solving the
antimicrobial menace globally especially in
developing world.
19
Recommendation
⬢ Round table discussion with policy
makers to prioritize AST for adequate
funding.
⬢ Training and capacity building for AST
scientist
⬢ Employment of up-to-date equipment
and technology to aid proper AST
⬢ Local production and standardization of
materials for AST
⬢ Quality control visits to laboratory to
ensure standard operating procedure
implementation for AST
⬢ Establishment of private or public
dedicated committee to encourage
effective AST to solve AMR

20. References
20
⬢ Bayot, M.L., Bragg, B.N. (2022). Antimicrobial susceptibility testing.
https://www.ncbi.nlm.nih.gov/books/NBK539714
⬢ Davey. R. (2021, November 16). History of antimicrobial discovery. https://www.news-
medical.net/amp/life-sciences/History-of-Antimicrobial-Discovery.aspx
⬢ Food and Agriculture Organization of the United Nations. (n.d). Antimicrobial
resistance. What is it. https://www.fao.org/antimicrobial-resistance/background/what-is-
it/en/
⬢ Prestinaci, F., Pezzotti, P., Pantosti, A. (2015). About Antimicrobial Resistance: a
global multifaceted phenomenon. Pathog Glob Health, 109(7),309-318.
⬢ World Health Organization. (2021). Antimicrobial Resistance.
https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance
⬢ Microbiology Resource Center - Truckee Meadows Community College. (n.d).
Antimicrobial Susceptibility Testing. https://www.tmcc.edu/microbiology-resource-
center/lab-protocols/antimicrobial-susceptibility-testing
⬢ Biomeriuex. (n.d). Antimicrobial resistance. https://amr.biomerieux.com/en/about-
amr/what-is-antibiotic-resistance/
⬢ Star Health. (2022, September15). Antibiotics – what are they, uses & side effects.
https://www.starhealth.in/blog/antibiotics

21. THANKS!
Any questions?
You can find me on LinkedIn at:
⬢ Hamidah (Haroon) Adekilekun
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