This document discusses various methods for detecting bacterial infections and testing antibiotic susceptibility. It begins by defining bacteria and describing common bacterial diseases. It then covers topics like Gram staining, common antibiotics and their mechanisms of action, antimicrobial resistance, and current methods for antibiotic susceptibility testing including disk diffusion, broth dilution, and E-tests. Faster methods are also discussed, such as using microdevices, nephelometry in VITEK 2, and molecular detection of 16S rRNA. The document provides details on how some commercial systems like BD Phoenix and Accelerate Diagnostics work to quickly identify bacteria and determine antibiotic minimum inhibitory concentrations.

1. 1
FIGHTING THE RESISTANCE
Presenter : Wanda Liu(劉鵬雯)
https://i.gifer.com/7fpi.gif

2. Bacterial infection diseases
2
Diarrhea Pneumonia Sepsis

3. What is bacteria?
3
• A single-cell living organism without nucleus.
Its size is around 2 μm.
• Shape include spheres, rods, spirals.
• Categorized as Gram-negative and Gram-
positive bacteria with Gram stain.
Gram-negative bacteria Gram-positive bacteria

4. Gram stain
• Gram stain is a laboratory procedure
used to detect the presence of
bacteria and sometimes fungi.
• It gives relatively quick results as to
whether bacteria or fungi are present
and, if so, the general type(s).
4
https://microbeonline.com/gram-staining-principle-procedure-results/
Gram stain reagent kit
https://www.bd.com/en-uk/products/diagnostics-
systems/stains-and-reagents/gram-stain
95 % alcohol

5. 5
The crystal violet- iodine complex
can bind with teichoic acids.

6. Antibiotics
• The chemical compound that kill
bacteria or inhibit its growth. They are
most important antimicrobial agent for
bacterial infection.
• Current antimicrobial susceptibility
testing method is slow which cause late
bacteria identification(2~3 days). A
Faster measurement is an urgent need.
• Empiric therapy based on suspicious
illness is adopted when the pathogen is
not identified, which caused serious
drug resistance problems.
6
https://www.onecallmedicalalert.com/blog/2013/05/top-
reasons-people-fall-in-their-home-medications/

7. Target strategies
• Target bacterial cell wall
• Penicillins, Cephalosporins
• Target cell membrane
• Polymyxins
• Interfere with essential bacterial enzymes
• Rifamycins, Lipiarmycins, Quinolones and Sulfonamides
• Protein synthesis inhibitors
• Macrolides, Lincosamides, and Tetracyclines 7
https://www.orthobullets.com/
basic-science/9059/antibiotic-
classification-and-mechanism

8. β-lactam antibiotics
• Contain a β-lactam ring in their molecular
structures. Includes penicillin derivatives (penams),
cephalosporins (cephems), monobactams, and
carbapenems.
• Binding penicillin binding proteins(PBPs) to inhibit
cell wall biosynthesis
• Bacteria often become resistant to β-lactam
antibiotics because of β-lactamase which can
break down the β-lactam ring structure.
• To overcome the resistance, β-lactam antibiotics
are often prescribed with β-lactamase inhibitors,
such as clavulanic acid.
8
Penicillins
Cephalosporins
https://www.wikiwand.com/en/%CE%92-lactam_antibiotic

9. β-lactam antibiotics
• Contain a β-lactam ring in their molecular
structures. Includes penicillin derivatives (penams),
cephalosporins (cephems), monobactams, and
carbapenems.
• Binding penicillin binding proteins(PBPs) to inhibit
cell wall biosynthesis
• Bacteria often become resistant to β-lactam
antibiotics because of β-lactamase which can
break down the β-lactam ring structure.
• To overcome the resistance, β-lactam antibiotics
are often prescribed with β-lactamase inhibitors,
such as clavulanic acid.
9
Penicillins
Cephalosporins
https://www.wikiwand.com/en/%CE%92-lactam_antibiotic
Clavulanic acid
https://www.wikiwand.com/en/Clavulanic_acid

10. Penicillin
• Discovered by Alexander
Fleming that Staphylococcus
aureus failed to growth in the
area contaminated by Penicillin
notatum.
• Penicillin and other β-lactam
antibiotics act by inhibiting
penicillin-binding proteins,
which normally catalyze cross-
linking of bacterial cell walls.
10
https://www.storyboardthat.com/storyboards/oliversmith/penicillin

11. Penicillin
• Discovered by Alexander Fleming
that Staphylococcus aureus failed
to growth in the area contaminated
by Penicillin notatum.
• Penicillin and other β-lactam
antibiotics act by inhibiting
penicillin-binding proteins, which
normally catalyze cross-linking of
bacterial cell walls.
11
https://www.wikiwand.com/en/Penicillin#/Mechanism_of_action

12. β-lactam antibiotics-Cephalosporins
• Originally derived from the fungus Acremonium. Together with cephamycins,
they constitute a subgroup of β-lactam antibiotics called cephems.
• Each newer generation has significantly greater Gram-negative antimicrobial
properties than the preceding generation
14
First generation Second generation Third generation Forth generation
Drugs Cefazolin, Cephalexin Cefuroxime, Cefmetazole Ceftriaxone, Cetazidime Cefepime, Cefpirome
G+
G-
Anaerobes 0 0
Effect Mainly against Gram
positive bacteria
Increase the activity
against Gram positive
bacteria
Significantly increase
Gram negative bacteria.
Some drugs have effect
on Pseudomonas
aeuriginosa
The most broad spectrum
of antimicrobial
3
3
3
3 3
3
4
4
4
2
1
Mandell Principles and Practice of Infectious Disease, 8th ; http://jerryljw.blogspot.com/2018/10/cephalosporins.html

13. Polymyxin B
• Used for G(-) bacterial infection.
• Composed of a number of related
compounds including polymyxins B1,
B1-I, B2, B3, and B6.
• Increase the bacterial outer membrane
permeability by binding negatively charged
lipopolysaccharide layer to prevent the
binding of positively charged amino
groups in the cyclic peptide portion
(this site normally is a binding site for
calcium and magnesium counter ions)
15
Polymyxin B1
http://www.enzolifesciences.com/alx-380-040/polymyxin-b-.-sulfate/
Polymyxin B2
https://medkoo.com/products/24195

14. Polymyxin B
• Used for G(-) bacterial infection.
• Composed of a number of related
compounds including polymyxins B1,
B1-I, B2, B3, and B6.
• Increase the bacterial outer membrane
permeability by binding negatively charged
lipopolysaccharide layer to prevent the
binding of positively charged amino
groups in the cyclic peptide portion
(this site normally is a binding site for
calcium and magnesium counter ions)
16
Polymyxin B1
http://www.enzolifesciences.com/alx-380-040/polymyxin-b-.-sulfate/
Polymyxin B2
https://medkoo.com/products/24195
The action mechanism of polymyxin B
(https://www.semanticscholar.org/paper/Antibacterial
-Mechanisms-of-Polymyxin-and-Bacterial-Yu-
Qin/888bf36dc61219252298a64b01cc727432714378)

15. Antimicrobial resistance (AMR)
• Antimicrobial resistance happens when micro-
organisms (such as bacteria, fungi, viruses, and
parasites) change when they are exposed to
antimicrobial drugs.
• Accelerating by the misuse and overuse of
antimicrobials with empiric therapies, which causes
threats towards treatments of common infectious
diseases and medical procedures like surgeries.
• Resistance in E. coli to one of the most widely used
medicines for the treatment of urinary tract
infections (fluoroquinolone antibiotics) is very
widespread. There are countries in many parts of
the world where this treatment is now ineffective in
more than half of patients.
(Ref : WHO 《antimicrobial resistance》)
18
https://equimanagement.com/articles/
antimicrobial-resistance-in-horses

16. 19
https://courses.lumenlearning.com/microbio
logy/chapter/drug-resistance/

17. 20
https://courses.lumenlearning.com/microbio
logy/chapter/drug-resistance/
• Aminoglycoside : resistance can occur
through enzymatic transfer of chemical
groups to the drug molecule, impairing the
binding of the drug to its bacterial target.
• β-lactams : enzymatic(β-lactamases)
hydrolysis of the β-lactam bond within the
β-lactam ring of the drug molecule.
• Rifampin : inactivation by glycosylation,
phosphorylation, or adenosine
diphosphate (ADP) ribosylation.

18. 21
• Carbapenem : Pseudomonas aeruginosa decreases its amount of its
OprD porin, which is the primary portal of entry for carbapenems
through the outer membrane of this pathogen.
• β-lactams, tetracyclines, and fluoroquinolones : produce efflux
pumps that actively transport an antimicrobial drug out of the cell and
prevent the accumulation of drug to a level that would be antibacterial.
https://courses.lumenlearning.com/microbio
logy/chapter/drug-resistance/

19. 22
https://courses.lumenlearning.com/microbio
logy/chapter/drug-resistance/
• β-lactam drugs :
1. Inhibit the binding with penicillin
binding protein(PBP), like
Streptococcus pneumonia.
2. Staphylococcus aureus develop
resistance to methicillin (MRSA)
through the acquisition of a new
low-affinity PBP, which provides
resistance to virtually all β-lactam
drugs, with the exception of the
newer fifth-generation cephalosporins
designed specifically to kill MRSA.
• Macrolides, tetracyclines, and
aminoglycosides : ribosome subunits
• Rifampin : RNA polymerase
• Fluoroquinolones : DNA gyrase

20. Antimicrobial susceptibility testing(AST)
•Disk diffusion method (established in 1940s)
A test of antibiotic sensitivity of bacteria, determined by the diameter
of inhibition zone.
23
Disk diffusion method

21. 24
1. Müller-Hinton agar:
• Beef Extract 2.00 gm
• Acid Hydrolysate of Casein 17.50 gm
• Starch 1.50 gm
• Agar 17.00 gm
• Distilled Water 1000 ml
• Final pH 7.3 ± 0.1 at 25ºC
2. Seed 0.5 McFarland (1.5x108 CFU/ml) of
bacterial suspension on Müller-Hinton agar.

22. •Broth dilution method
• Determines with turbidity
• The reference method for Minimum
inhibitory concentration(MIC) test.
• Müller-Hinton broth are suggested in
CLSI(The U.S. Clinical and Laboratory
Standards Institute) guidelines.
• The method includes :
• Broth macrodilution method
• Broth microdilution method
25
Resistant Sensitive
Antimicrobial susceptibility testing(AST)

23. 26
Broth dilution
method

24. 27
Broth macrodilution method

25. 28
Broth microdilution method

26. Minimum inhibitory concentration(MIC) test
29
• The lowest concentration (µg/mL) of a antibiotic,
which prevents visible growth of bacterium. It’s
important to identify the varying levels of resistance
of different serotypes of bacteria to antimicrobials.
• Results have been graded into susceptible,
intermediate, or resistant to a particular
antimicrobial by using a breakpoint. Breakpoints are
agreed upon values, published in guidelines of a
reference body, such as :
• The U.S. Clinical and Laboratory Standards Institute (CLSI)
• The British Society for Antimicrobial Chemotherapy (BSAC)
• The European Committee on Antimicrobial Susceptibility
Testing (EUCAST)
http://www.esuppliersindia.com/lingam-
microbiological-laboratory/mic-test-
strip-microbiological-laboratory-631502-
testing-service-pr4607215-sFP-swf.html
E-test

27. •E-test (Epsilometer test)
1. The quantitative method to determine the
MIC by using a inert and non porous plastic
reagent strip with a gradient of antibiotic,
covering a continuous concentration range.
2. Manufactured by bioMérieux, and it is the
most common method used in healthcare
settings help physician in the treatments.
3. When an even lawn of growth is distinctly
visible, the MIC value can be read where
the edge of the inhibition ellipse intersects
the side of the strip.
30
https://www.wikiwand.com/en/Etest
Antimicrobial susceptibility testing(AST)

28. •E-test(Epsilometer test)
1. The quantitative method to determine the
MIC by using a inert and non porous plastic
reagent strip with a gradient of antibiotic,
covering a continuous concentration range.
2. Manufactured by bioMérieux, and it is the
most common method used in healthcare
settings help physician in the treatments.
3. When an even lawn of growth is distinctly
visible, the MIC value can be read where the
edge of the inhibition ellipse intersects the
side of the strip. 31
Zone of inhibition
Bacteria growth
MIC result
Ceftazidime
Antimicrobial susceptibility testing(AST)

29. 32
Is there any faster AST method?

30. Electrochemical microdevice for determination of the minimum inhibitory concentration of antibiotics. (A) Image of the device.
(B) Schematic of the device. (C) Culture chamber (0.4 μL). (D) Three-electrode system for impedance measurement in each
culture chamber. Working electrode (W.E.), Ag; auxiliary electrode (A.E.), Ag; reference electrode (R.E.), Ag/AgCl.
Analyst, 2018, 143, 396
33

31. Fig. Impedance measurements as a function of frequency and culture time. (A) Dependence of impedance
on frequency. Samples containing E. coli cells were incubated for 6, 12, and 18 h. The applied potential
was −0.8 V vs. Ag/AgCl. (B) Changes in the impedance when E. coli cells were cultured in three different
chambers without antibiotics. Applied potential and frequency were −0.8 V vs. Ag/AgCl and 1 kHz.
Analyst, 2018, 143, 396
34

32. 35
VITEK 2

33. Detection of antibiotics resistance
• VITEK 2 - nephelometry
36
BMGLABTECH 《Monitoring of microbial growth curves by laser nephelometry》
VIEK 2

34. VITEK 2
2
1
3
Cards : ID cards, AST cards
64 Testing wells
Air draw hole(vacuum
pumping to help sample
moving)
Channels
4 Sample inlet
37

35. Prepare 0.5 McF
sample in advance
Sample inlet
38

36. 39
BD Phoenix

37. 40
BD Phoenix

38. BD Phoenix
1 Antimicrobial susceptibility
testing, 85 wells
2 Identification, 51 wells
Testing chips
Reagents
41

39. 1
2
3
4
Dip sample colony and mix with ID
reagent and adjust the concentration
at 0.5 McF
Using pipet to add a little the
bacteria fluid into AST reagent
Add indicator into AST reagent
Add ID reagent and AST reagent
into the testing chip.
42

40. 43
Accelerate diagnostics

41. 1
3
Using blood culture sample
Add processed sample
into the testing chip
2 Separate bacteria from other
impurities
Accelerate diagnostics
44

42. • Bacteria purification
• Using electrical field
effect to separate
bacteria from other
impurities
Electrofiltration
45

43. Accelerate diagnostics: The Testing Chip
46
Identification AST

44. 47
Testing agents : E. coli
PIP-TAZO : Piperacillin/tazobactam

45. Molecular detection-
16S ribosomal RNA (rRNA)
49
Structure of 16S ribosomal RNA
• 16S rRNA (16S ribosomal RNA)
Contains highly conserved primer binding
sites with hypervariable regions that provide
species-specific signature sequences useful
for identification of bacteria.

46. 50
http://genefluidics.com/img_0040-4/
Genefluidics
Procedure assumption :
After forepreparation and
incubation with antibiotics…

47. 51
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2006, p. 561–570
Reading machine
Clinical urine
specimen containg
K.pneumonia

48. Genefluidics
54
http://genefluidics.com/ast/antimicrobial-susceptibility-testing/

49. 55
J Urol. 2011 January ; 185(1): 148–
153. doi:10.1016/j.juro.2010.09.022
AXO : Ceftriazone
FEP : Cefepime

50. Detection of antibiotics resistance
56
ARG-ANNOT database
Identify antibiotic resistance-
coding genes
(Ref : Antimicrob Agents Chemother 2014; 58:212–20.)
Real-time PCR
Targeting antibiotic resistance genes of
clinical relevance
Exp. Identify OXA-48 that codes carbapenem
resistance in Enterobacteriaceae. (腸桿菌科)
(Ref : J Antimicrob Chemother 2004; 54:538–41.)

51. 57
Smarticles
https://www.labnetwork.com.br/noticias/roche-compra-empresa-
com-foco-em-microbiologia-fundada-por-ex-bolsista-brasileiro/
https://healthdocbox.com/Cance
r/75495113-Committed-to-
innovation-and-growth.html

52. 58
https://diagnostics.roche.com/global/en/article-listing/smarticles-technology.html
Identification

53. 59
https://diagnostics.roche.com/global/en/article-listing/smarticles-technology.html
AST

54. Developing directions
60
Colony formation Bacteria count Bacteria shape
Genomic analysis Enzymatic activity

55. 61