If antibiotics loose their effectiveness, the ability to treat many diseases will be lost. It is therefore only necessary that they be used appropriate in the right conditions. Microbiologists must ensure to adhere to Standard Operating Procedures in Antibiotic Susceptibility Testing.

1. CAUSES OF ANTIBIOTIC
RESISTANCE IN
SUSCEPTIBILTY TESTING: IN
MEDICAL MICROBIOLOGY
BY
MLS ABANG, LARRY BLESSING
SUPERVISED BY MLS C.F.N. ONUOHA
13 FEBRUARY, 2024

2. OUTLINE
Introduction
Bacteria
Antibiotics
Antimicrobial resistance
Causes of antimicrobial resistance
Susceptibility testing
Mechanism of antimicrobial resistance
Microscopy, culture and sensitivity
2

3. OUTLINE
Importance of antibiotic susceptibility testing
Antibiotic susceptibility testing methods
Causes of resistance in susceptibility testing
Case studies
Conclusion
Recommendations
References
3

4. INTRODUCTION
Microbes are minute, unicellular organisms that cannot be seen with the naked
eyes.
While are some are useful to the human body, others are harmful to health.
These harmful organisms are called ‘pathogens’.
Still, some of these microbes are typically ‘non-pathogenic’ and would act as
pathogens in some situations, causing disease.
The disease causing microbes include bacteria, viruses, protozoa, fungi, and
helminths.
4

5. INTRODUCTION (cont.)
Antimicrobial agents include naturally occurring antibiotics, derivatives of
naturally occurring antibiotics and chemical antimicrobial compounds.
The term “antibiotics” is used to describe antimicrobial agents, against bacteria,
that can be used to treat infection.
Not all antimicrobials are completely non-toxic. They may show a selective
toxicity in the treatment of microbial diseases.
5

6. BACTERIA
 Bacteria (singular-bacterium) are unicellular microorganisms.
 They are part of a human’s microbiome, existing in the digestive system, on the skin,
etc.
 In cases of invasion, they are able to reproduce quickly and release toxins that can
cause infection.
 Examples of bacteria include Streptococcus, Staphylococcus, Eschericia coli, etc.
 These are the most common bacteria that cause septicemia and subsequently, sepsis.
(Raza et al., 2021)
6

7. ANTIBIOTICS
 Antibiotics can be grouped by their mode of action
i. Inhibitors of bacterial cell wall synthesis e.g. penicillin, cephalosporin,
carbapenems.
ii. Inhibitors of protein synthesis e.g. tetracyclines, macrolides
iii. Inhibitors on nucleic acid synthesis e.g. quinolones, metronidazole,
sulphonamides.
 They may act either as being bactericidal or bacteriostatic
(Cheesebrough, 2006)
7

8. ANTIMICROBIAL RESISTANCE
 The WHO defines Antimicrobial resistance (AMR) as a microorganism’s resistance to
an antimicrobial drug that was once able to treat an infection by that organism.
 It is important to note that a person does not become resistant to antibiotics.
Resistance IS a property of microbes, not humans or any other organism infected by
a microbe.
 Antibiotic resistance is a subset of AMR to bacteria that applies specifically to
bacteria that become resistant to antibiotics.
(Bagul & Sivakumar, 2016)
8

9. CAUSES OF ANTIMICROBIAL RESISTANCE
Overuse/misuse of antimicrobials: this leads to microbes either evolving a
defense against the drugs used to treat them or certain strains of microbes that
have a natural resistance to antimicrobials becoming much more prevalent than
those that are susceptible.
i. Over-prescription/inappropriate prescription of antibiotics
ii. Incomplete dosage: many individuals stop taking antibiotics when they begin
to feel better.
(Bagul & Sivakumar, 2016)
9

10. CAUSES OF AMR (CONT.)
Natural selection: organisms that are able to adapt to their environment, survive and
continue to produce offspring.
i. As a result, the type of organisms that are able to survive over time with continued
exposure to antibiotics will naturally become prevalent in the environment rendering the
susceptible ones obsolete.
ii. There could also be a horizontal gene transfer. Here, once a gene for resistance to an
antibiotic appears in an antimicrobial community, it can spread to other microbes in that
community, potentially moving from a non-disease causing microbe to a disease causing
microbe.
(Bagul & Sivakumar, 2016)
10

11. CAUSES OF AMR (CONT.)
 Self medication: this is the taking of medicines on one’s own initiative or on the suggestion of
another person, who is not a certified medical professional. This is an unsuitable way of drug use
but a common practice in resource-constrained countries. This may be a resort in the following
cases:
i. In cases of lockdowns
ii. Unavailability of health professionals
iii. Limited amount of time
iv. Financial constraints
(Bagul & Sivakumar, 2016)
11

12. CAUSES OF AMR (CONT.)
 Clinical misuse: a research by Ventola (2015) showed that the indication for
treatment with antibiotics, choice of agent to be used, and duration of therapy was
incorrect in up to 50% of the cases studied.
 Another study showed that about 1 in 3 prescriptions are unnecessary
 Oftentimes, physicians underestimate the impact that their own prescribing habits
have on microbial resistance.
 It could also be that some physicians may be overly cautious and prescribe
antibiotics for both medical or legal reasons, even when clinical indications are not
always confirmed.
12

13. SUSCEPTIBILITY TESTING
Antibiotic Susceptibility Testing (AST), also known as Antibiotic Sensitivity Testing, is
the measurement of bacteria to antibiotics. This is done because bacteria may have
resistance to some antibiotics.
It allows a clinician’s choice of drugs from empiric therapy (where an antibiotic is
selected based on clinical suspicion about the site of infection and common causative
bacteria. to directed therapy), to directed therapy, where a choice of antibiotic is a based
on knowledge of the organism and its sensitivities.
One of the significant roles of the medical microbiology laboratory is the performance
of antimicrobial susceptibility testing of various bacterial isolates.
( Leeka, 2011)
13

14. MECHANISM OF ANTIMICROBIAL
RESISTANCE
Microorganisms become resistant to antimicrobials through several ways which
include:
i. Production of beta-lactamase enzymes that destroy the beta-lactam ring of
penicillin and cephalosporins
ii. Altering permeability of outer membrane of bacterial cell wall
iii. Mutation
iv. Bacterial efflux pump to eject antibiotics
v. Production of metabolic by-passes to antimicrobial action
(Bagul & Sivakumar, 2016)
14

15. MICROSCOPY, CULTURE AND SENSITIVITY
(M/C/S)
 Microscopy, culture and sensitivity is the term used when sending certain
samples to the microbiology laboratory
After receipt of the patient’s sample collected on Day 0, the clinical microbiologist
MUST isolate the potential pathogen by streaking the sample on selective culture
media and incubating the inoculated media overnight (or longer) to enable
growth.
 Microscopy is then done in order to enable a quick initial report
(Syal et al., 2017)
15

16. MICROSCOPY, CULTURE AND SENSITIVITY
(M/C/S)
 From the primary growth plate, (Day 1) isolated colonies are obtained by sub-culture.
 Once the isolated pathogens are available (Day 2), the bacteria inoculum is prepared and
standardized.
 AST may then be performed .
 The minimum inhibitory concentration (MIC) is the lowest concentration of antibiotic
required to prevent bacteria growth and it is used to determine if the infective pathogen is
susceptible or resistant.
(Syal et al., 2017)
16

17. MICROSCOPY, CULTURE AND SENSITIVITY
(M/C/S)
It is also important to note the clinical breakpoint. A breakpoint is the
concentration of antibiotics that enable the interpretation as susceptible,
intermediate or resistant.
The breakpoint is different for different antibiotics and bacteria
MIC has to be less than or equal to the breakdown for it to be termed susceptible.
(Syal et al., 2017)
17

18. IMPORTANCE OF ANTIBIOTIC
SUSCEPTIBILITY TESTING (AST)
i. In the determination of the effectiveness of antibiotic therapy against a
bacterial infection.
ii. In the control of the use of antibiotics in clinical use.
iii. Assistance in drug choice for treatment of infections by clinicians.
iv. Aiding the local pattern of drug prescription.
v. To reveal the changing trends in local isolates.
(Bagul & Sivakumar, 2016)
18

19. ANTIBIOTIC SUSCEPTIBILITY TESTING
METHODS
Qualitative method
i. Disk diffusion test (Kirby-Bauer disk method)
ii. Well diffusion method
iii. Stokes method
Quantitative method
i. Tube or macro broth dilution
ii. Agar diffusion
iii. E-test
19

20. 20

21. 21
E-test method
Pharmscholars.com

22. 21
ACNHA LAB

23. KIRBY BAUER DISC METHOD:
REQUIREMENTS
Mueller Hinton agar: which is prepared and sterilized according to
manufacturer’s instruction. The pH oshould be 7.2-7.4, pour in a 90mm diameter
sterile Petri dish to a depth of 4mm (about 25mL/plate).
NOTE: if medium is too thin, zones will be falsely large and if too thin, zones will
be too falsely small
(Cheesebrough, 2006)
23

24. KIRBY BAUER DISC METHOD:
REQUIREMENTS
Antibiotic discs: which are chosen based on the specimen,
pathogen, available discs, and the prescribing policies.
Turbidity standard equivalent to MacFarland 0.5: this is a standard
against which the turbidity of test and control inocula can be
compared
Control strains
(Cheesbrough, 2006)
24

25. KIRBY BAUER DISC METHOD: PROCEDURE
Using a sterile wire loop, touch, 3-5 well isolated colonies of similar appearance
to the test organism and emulsify in 3-4mL 0f sterile physiological saline
In good lighting, match the turbidity of suspension to the standard.
With a sterile swab stick, inoculate MH agar plate. Ensure to maintain an even
streak over the plate.
With lid in cover, allow plate to dry for about 3-5 minute
(Cheesebrough, 2006)
25

26. KIRBY BAUER DISC METHOD: PROCEDURE
(CONT)
Using sterile forceps, place the appropriate disc on plate. Discs should be about 15mm from
the edge of plate and no closer than 25mm from disc to disc. No more than 6 discs on a plate.
Disc should be lightly pressed down to ensure contact with agar.
Within 30 minutes, invert plates and incubate at 35-370C for 18-24 hours
Examine the control and test plates. Use a ruler on the underside and measure each zone of
inhibition.
(Cheesebrough, 2006)
26

27. KIRBY BAUER DISC METHOD (CONT)
This area is the zone of inhibition (ZOI) and it is directly proportional to the
sensitivity of the isolate and to the diffusion rate of antibiotic onto the agar
medium.
The ZOI is measured in millimeters by either measuring:
i. Radius: in cases where there is an unclear growth or a merging of zones. It is
multiplied by 2.
ii. Diameter: the entire length of the zone is measured and the diameter of the
disc area (5-6mm) is subtracted.
(Syal, 2017)
27

28. 28

29. 29
Pharmscholars.co
m

30. KIRBY BAUER DISC METHOD (CONT)
The result of the test should be interpreted by using the criteria published by Clinical and
Laboratory Standard Institute (CLSA).
Results should be represented as
i. Susceptible: where isolates are inhibited by the usually recommended dosage of
antimicrobial agents
ii. Intermediate: where responses are lower than that of susceptible pathogens. Larger doses
are usually required.
iii. Resistant: where isolates are uninhibited by the usually achievable concentrations of
antibiotics.
(Bagul & Sivakumar, 2017)
30

31. CAUSES OF RESISTANCE IN SUSCEPTIBILITY
TESTING
Wrong sample collection
Time between sample collection and culturing
Density of the inoculum
Disk application time
Temperature of incubation
(Bagul & Sivakumar, 2017)
31

32. CAUSES OF RESISTANCE IN SUSCEPTIBILITY
TESTING (CONT)
Potency of drug
Inappropriate storage conditions
pH of agar medium
Moisture on surface of medium
Drug misuse
Long admittance in the hospital
(Bagul & Sivakumar, 2017)
32

33. CASE STUDY ONE
A 29-year old female patient, Faith Fergusson (3024079), who is being managed at
this facility for a seizure disorder. Prior to laboratory investigations, she had ben
placed on ceftriaxone, metronidazole, ciprofloxacin. A HVS M/C/S was done and
the isolated organism (Pseudomonas spp) was resistant to wide range of antibiotics
including ciprofloxacin.
A wound swab M/C/S was done and isolated organism, which was sensitive to
only Imipenem(++). The bacterial was resistant to a wide range of antibiotics
including ceftriaxone and ciprofloxacin.
This sensitivities were done as at 16 January, 2024
33

34. As at the 5th of February, 2024, a wound swab was done again. This time, isolated
organism(s) (E. coli, ?Pseudomonas spp), were resistant to all antibiotics including
imipenem to which it was formerly susceptible to, gentamicin amoxiclav,
meropenem which were her medications at the time. Discuss.
34

35. CASE STUDY TWO
A 77-year old male patient, Bako Karma (3024782) who presented at this facility
with a request of Urine M/C/S. He is being managed for BPH at National Hospital
and had currently being on medications for UTI prior to his visit.
Before a readiness of his results, this patient was already prescribed levofloxacin
The sensitivity testing showed that he was resistant to all antibiotics which were
used for the isolated organism (Klebsiella spp) including levofloxacin. Discuss.
35

36. CONCLUSION
Antimicrobial resistance is a threat to public health. Accurate and rapid detection
of resistance to antimicrobial drugs and subsequent appropriate treatment are
essential for controlling the emergence and spread of AMR.
36

37. RECOMMENDATIONS
Laboratory scientists must endeavor to follow standard operating procedures
on susceptibility testing.
Laboratory scientists should be well versed in the use of antibiotics specific to
suspected pathogen and site of sample collection
There should be a knowledge of the locally available antibiotics.
37

38. RECOMMENDATIONS
Medical laboratory scientists and clinicians MUST set aside inter-
professional rivalry and learn to work together for the greater good
of the patients.
Equipment used must be properly maintained and serviced.
Laboratories should work towards having an uninterrupted power
supply.
38

39. REFERENCES
ACNHA Laboratories. ACNHA/DAM 503/01Aii
Bagul, Uddhav & Sivakumar, Sivagurunathan. (2016). ANTIBIOTIC SUSCEPTIBILITY TESTING: A REVIEW ON
CURRENT PRACTICES. International Journal of Pharmacy. 6. 11-17.
Cheesebrough, M. (2006). Antibiotic Susceptibility Testing. District Laboratory Practices in Tropical Countries.
Chapter 7 pp 132-143
Gajic I, Kabic J, Kekic D, Jovicevic M, Milenkovic M, Mitic Culafic D, Trudic A, Ranin L, Opavski N. Antimicrobial
Susceptibility Testing: A Comprehensive Review of Currently Used Methods. Antibiotics (Basel). 2022 Mar
23;11(4):427. doi: 10.3390/antibiotics11040427. PMID: 35453179; PMCID: PMC9024665.
Leekha S, Terrell CL, Edson RS (February 2011). "General principles of antimicrobial therapy". Mayo Clinic
Proceedings. 86 (2): 156–67. doi:10.4065/mcp.2010.0639. PMC 3031442. PMID 21282489
Pharmscholars.com
39

40. Raza S, Matuła K, Karoń S, Paczesny J. Resistance and Adaptation of Bacteria to Non-Antibiotic Antibacterial
Agents: Physical Stressors, Nanoparticles, and Bacteriophages. Antibiotics. 2021; 10(4):435.
https://doi.org/10.3390/antibiotics1004043
Syal K, Mo M, Yu H, Iriya R, Jing W, Guodong S, Wang S, Grys TE, Haydel SE, Tao N. Current and emerging
techniques for antibiotic susceptibility tests. Theranostics. 2017 Apr 10;7(7):1795-1805. doi: 10.7150/thno.19217.
PMID: 28638468; PMCID: PMC5479269.
Ventola CL (April 2015). "The antibiotic resistance crisis: part 1: causes and threats". P & T. 40 (4): 277–
83. PMC 4378521. PMID 25859123
40

41. 41