Bacterial Resistance Mechanism, Biofilm Formation

1. Mechanisms of Antibiotic
Resistance In Bacteria &
Biofilm
PRESENTED BY
PRINCE
M.PHARM. 2nd SEM
DEPARTMENT OF PHARMACOLOGY
FACULTY OF PHARMACY
JAMIA HAMDARD

2. Index
I. Overview.
II. Origin of resistance
III. Major mechanisms of resistance
IV. Factors that promote bacterial resistance
V. Consequence of antibiotics resistance
VI. New trends for overcoming bacterial
resistance
VII. Biofilm

3. Definitions
Antibacterial Resistance
• It’s define as resistance of a microorganism to an
antimicrobial medicine to which it was originally
sensitive.
• Resistant organisms (they include bacteria, fungi, viruses and
some parasites) are able to withstand attack by antimicrobial
medicines, such as antibiotics, antifungals, antivirals, and
antimalarial that standard treatments become ineffective and
infections persist increasing risk of spread to others. The
evolution of resistant strains is a natural phenomenon that
happens when microorganisms are exposed to antimicrobial
drugs, and resistant traits can be exchanged between certain
types of bacteria.(WHO 2013)

4. (cont.)
Multi-drug resistance (MDR)
• Is defined as having acquired non-susceptibility
to more than one antimicrobial categories.
Pandrug-resistant (PDR)
• Is defined as non-susceptibility to all agents in
all antimicrobial categories.

5. ORIGIN OF RESISTANCE
Bacterial resistance to antimicrobial agents may
be intrinsic or acquired, intrinsic resistance as
resistance of Mycoplasma species to B-lactams
antibiotic, due to it’s lack of cell wall and
pleomorphic characters.
And acquired resistance is arise from de novo
mutation of DNA sequence or by horizontal gene
transfer by different mechanisms (transformation,
transduction and conjugation ).

6. Origin of resistance
Intrinsic resistance(IR)
is that type of resistance which is naturally coded and
expressed by all (or almost all) strains of that particular
bacterial species. An example of intrinsic resistance is the
natural resistance of anaerobes to aminoglycosides and
Gram-negative bacteria against Vancomycin.
 the resistant genes are maintained in nature because of
the presence of antibiotics producing bacteria in soil. These
antibiotics act on other bacterial species other than the
producer bacteria, There has to be a mechanism of
protection in the host bacteria against the antibiotics that it
produces, which could be the source of genes encoding

7. (cont.)
 is the innate ability of a bacterial species to resist activity of
a particular antimicrobial agent through its inherent
structural or functional characteristics, which allow
tolerance of a particular drug or antimicrobial class. This
can also be called “insensitivity” since it occurs in
organisms that have never been susceptible to that
particular drug. Such natural insensitivity can be due to:
I. lack of affinity of the drug for the bacterial target.
II. Inaccessibility of the drug into the bacterial cell.
III. Extrusion of the drug by chromosomally encoded
active exporters.
IV. Innate production of enzymes that inactivate the
drug.

8. (cont.)
Acquired resistance(AR)
Acquired resistance is said to occur when a particular
microorganism obtains the ability to resist the activity of a
particular antimicrobial agent to which it was previously
susceptible.
By mutation
By horizontal gene transfer
1. Mutation
It’s define as permanent change in the sequence of DNA
nucleotide of gene. This change can take place either by
alteration, loss or gain of the nucleotide.
 Types
1. Spontaneous mutation ( occurs by natural physical agents as
HEAT and IRRADIATION , in which energize DNA nucleotide so
that subsequent intra-molecular rearrangement of bases lead to
incorrect base –pairing and ultimately mutation).
2. Induced mutation(occurs by intentional treatment of the cell with

10. (cont.)
2- Horizontal gene transfer(HGT)
It’s recombination between two genetically different DNA
molecules, then the resistance is acquired. Acquisition of
foreign genetic elements in prokaryotes may occur by
three main mechanisms.
I. TRANSFORMATION → direct passage of free DNA (naked)
from one cell to another. The receiving bacteria then simply
introduce the free DNA in to their cytoplasm and then
incorporate it to their own DNA.
II. TRANSDUCTION → transfer of genetic element by mean of
vector (usually virus) called bacteriophage.
III. CONJUGATION→it’s the most important and most common
mechanism of gen transfer, this mechanism is mediated by
plasmid (bacteria containing plasmid called F positive. But the
other cell is called F negative.

11. (cont.)
Transposon
 It’s a mobile genetic element involved in horizontal gen transfer.
 Have the ability to move from place to place on the chromosome
and in to and out plasmid.
 Types:
1- Replicative → it's leave a copy of itself at the original
site.
2- Non replicative → it's not leave a copy of itself at the
original site.
N.B. transposon can enter the functional gene
 Size about 5 kilobases.
 Two enzyme are involved in transposition process
1-Transposase
2-Resolvase

14. Major biological mechanisms of
antimicrobial resistance
 Decreased uptake(impermeability)
and increased efflux of drug from the
microbial cell
 Drug Inactivation
 Change in shape of receptor/decrease
in permeability
 Use of alternative metabolic pathways

15. (cont.)
I. Decreased uptake(impermeability) and increased efflux of
drug from the microbial cell: -Efflux systems function via an
energy-dependent mechanism (active transport) to pump
out drug as well as unwanted toxic substances through
specific efflux pumps, this involves the involvement of P-gp
which is a type of efflux ABC channel

16. (cont.)
 E.g. P. aeruginosa and E.coli are containing proton-
dependant efflux pump which expel the drug outside the
cell.
 Examples
Tetracyclin resistance byTetA,B and k gene mediated efflux pump.
Fluroquinolon resistance by decrease uptake
Vancomycin resistance By increase thickness of bacterial cell wall,
so decrease uptake.
 EFFLUX
 AND
 IMMPERMEABILITY

17. (cont.)
II. Drug Inactivation
Some antibioitics are inactivated by some
enzymes present in bacterial body like
pencillinase, rendering the drug inactive and this
antibiotic further will not have any effect on
bacteria, making them resistant.

19. (cont.)
III. Change in shape of receptor/decrease in
permeability.
 Mutation can change the shape of receptor and
causes the change in conformation making the drug
unable to bind completely to receptor, so no signal
will be produce this is also a type of antibiotic
resistance mechanism in bacteria.
 E.g.

20. (cont.)
IV. Use of alternative metabolic pathways.
 Some drugs block the usual metabolic pathway,
bacteria circumvent this condition by using an
alternative, unblocked pathway that produces the
required product
Example: Sulphonamides

21. (cont.)
 Alteration in gene and gene products : There are
various genes and gene products which are present in
bacterial cell code for various functions (like enzyme
synthesis, cell wall formation etc.). Sometimes there is
change in these gene which lead to non generation of active
metabolite of drugs that could inhibit the bacterial growth.
 Examples: Isoniazid resistance due to alteration in inhA or
kasA gene, KatG gene.
 Pyrazinamide resistance due to alteration in pncA gene
 Ethambutol resistance due to alteration in embB gene

23. Biofilm
A coherent cluster of bacterial cells embedded in a
matrix, which is more tolerant of most
antimicrobials and host defences compared with
planktonic bacterial cells.
Biofilm formation can result in tolerance of bacteria to
very high concentrations of multiple antibiotics,
resulting in chronic infections despite antibiotic
treatment.

24. Quorum Sensing
• It is a density-dependent trait involves
bacterial cell-to-cell communication,
known as quorum sensing (QS),
where the ‘quorum’ refers to the
minimum number of bacteria
aggregated within a specific volume
that is required to make a ‘decision’ to
switch on the gene expression of QS-
controlled genes

25. Diagram representing quoram sensing mechanism

26.  Steps of biofilm formation
I. Formation of conditioning biofilm.
II. Initial attachment.
III. Irreversible attachment and synthesis
and secretion of a matrix consisting of
extracellular polymeric substance
(EPS). This EPS matrix accumulates
and eventually surrounds the population
of bacterial cells
IV. Biofilm growing.
V. Detachment.
VI. Formation of a new conditioning biofilm
in other site in host.

28. Why Bacteria Form Biofilm
?

29. Role of Extracellular polymeric substance in
resistanse
I. Act as selective permeable for diffusion of oxygen and
nutrients.
II. Decrease diffusion of antibacterial agent to bacterial
population, so concentration not reach to MIC due to:
 Small pores of EPS.
 The negative charge of the EPS matrix also traps
antibiotic molecules before they can affect the bacterial
cells
 Enzymes within the EPS matrix also covalently modify
antibiotic molecules, thereby inactivating their
antimicrobial activity.

30. Biofilm Resistance
Treatment
 QS inhibitors: Usnic acid, a lichen
metabolite, possesses inhibitory
activity against bacterial and fungal
biofilms via QS interference. QS
inhibitors can increase the
susceptibility of biofilms to antibiotics.
QS Inhibitors are generally regarded
as safe in humans.

31.  Biofilm-Disrupting Enzymes: Enzymes,
like DNase I, α-amylase and DspB are
biofilm-dispersing agents that degrade the
biofilm matrix, permitting increased
penetration of antibiotics. DNase I cleavage
of extracellular DNA leads to alterations in
biofilm architecture, which permits increased
antibiotic penetration
 Low-frequency ultrasound: treatment in
combination with antibiotics is promising for
biofilm removal. Ultrasound facilitates
transport of antibiotics across biofilms, and
increases sensitivity of biofilm-growing
bacteria to antibiotics. It has been used as a
treatment for chronic rhinosinusitis.

32.  Flavonoids :The anticancer,
antioxidant, and anti-inflammatory
effects of flavonoids are well
established. Yet, their biofilm
disrupting function is practically
unknown. Flavonoids appear to
suppress the formation of biofilms via
a non-specific QS inhibition. The
flavonoid phloretin inhibited biofilm
formation in E. coli.

33.  Treatment by shock waves:-To determine
whether this enhanced dispersal led to
increased sensitivity of the biofilm to antibiotic
treatment, biofilms in microfuge tubes and
catheters were exposed to shock waves,
incubated with 4 μ g/ml ciprofloxacin for 6 h,
the biofilm fully dispersed using a bath
sonicator, and plated for viable count.The
results showed that, while biofilms were
structurally resistant to the antibiotic, the
bacteria became susceptible to antibiotic
after treatment with the shock waves . The
use of the shock wave increased the
biofilmncommunity’s sensitivity to antibiotic by
100 to > 1,000-fold.

34. Antibiotic sensitivity of bacteria released by shock wave treatment. Catheter sections with biofilms
of P. aeruginosa or S. aureus formed in bovine (a) or human (b) urine were washed and placed in PBS.
After
shock wave exposure, the PBS was plated to check the release of the bacteria from the biofilm.

35. Factors that promote bacterial resistance
Suboptimal use of antimicrobials for prophylaxis
and treatment of infection.
Prolonged hospitalization, increased number and
duration of intensive care-unit stays, multiple
comorbidities in hospitalized patient.
 Increased use of invasive devices and catheters.
 Ineffective infection-control practices, transfer of
colonized patients from hospital to hospital
 Antibiotic used in agriculture and household.
 Increasing national and international travel.
 Lack of education and poverty.

36. Consequence of antibacterial resistance

38. New trends for overcoming bacterial
resistance
Due to global emergence of antibacterial resistance,
scientists are introduce a new strategies to overcome
resistance.
Many of this strategies are
I. Plant compounds with resistance modifying activities.
II. Nanotechnology as a therapeutic tool to combat
microbial resistance.

39. I. Some antibiotic resistance modifying compounds from
plants
REFERANCEANTIBIOTIC
POTENTIATED
PLANT SOURCECOMPOUND
Smith et al. (2007)Oxacillin,
Tetracycline,
Norfloxacin
Tetracycline
Chamaecyparis
lawsoniana
Ferruginol
5-Epipisiferol
Marquez et al.
(2005)
Ciprofloxacin,
Norfloxacin,
Pefloxacin,
Acriflavine and
Ethidium bromide
Jatropha elliptica2,6-dimethyl-4-
phenylpyridine-
3,5-dicarboxylic
acid diethyl ester
Oluwatuyi et al.ErythromycinRosmarinusCarnosic acid

40. II. Nanotechnology as a therapeutic tool to
combat microbial resistance.
Use of nanoparticles is among the most promising
strategies to overcome microbial drug resistance.
Example
Nanoparticles with multiple simultaneous mechanisms of
action against microbes
Nitric oxide-releasing nanoparticles (NO NPs).
Chitosan-containing nanoparticles (chitosan NPs).
Metal-containing nanoparticles.
Nanoparticles that target antimicrobial agents to the site
of infection.
Liposomes nano-particles.
Dendrimers.

41. Finally

42. References
 Bartley J, Young D. Ultrasound as a treatment for
chronic rhinosinusitis. Med
Hypotheses 2009;73:15–7.
 Abraham KP., Sreenivas J, Venkateswarulu TC, et
al. Investigation of the potential antibiofilm activities
of plant extracts. International Int J Pharm Pharm
Sci 2012;4:282-5.
 Divya Prakash Gnanadhas et.al.Successful
treatment of biofilm infections using shock waves
combined with antibiotic therapy. Nature
Publications.5:17440 | DOI: 10.1038/srep17440

43.  Kohler T, Guanella R, Carlet J, van DC. Quorum
sensing-dependent virulence during Pseudomonas
aeruginosa colonisation and pneumonia in
mechanically ventilated patients. Thorax
2010;65:703–10
 Costerton JW, Post JC, Ehrlich GD, Hu FZ, Kreft R,
Nistico L, et al. New methods forthe detection of
orthopedic and other biofilm infections. FEMS
Immunol Med Microbiol 2011;Mar;61(2):133–40
 Stapper AP, Narasimhan G, Ohman DE, Barakat J,
Hentzer M, Molin S, et al. Alginate production
affects Pseudomonas aeruginosa biofilm
development and architecture, but is not essential
for biofilm formation. J Med Microbiol 2004;53(Pt
7):679–90

44.  Essential Of Medical Pharmacology 7th edition, by
Tripathi KD, published by Jaypee Brothers medical
publishers,2013, page no. 740, 765-69.