bacterial resistance

1. Bacterial resistance
By:
Mohammed
Fawzy
Microbiology
lab at NODCAR

3. Agenda
I. Overview.
II. Origin of resistance
III. Major mechanisms of resistance
IV. Factors that promote bacterial resistance
V. Antibacterial in food and animal industries
VI. Consequence of antibiotics resistance
VII.New trends for overcoming bacterial resistance
VIII.Questions

4. overview
Worldwide, antibacterial resistance has increased dramatically over the
past few years and is currently recognized as a major medical challenge
in most healthcare settings.
After the discovery of penicillin in 1928, a number of treatment failures
and occurrence of some bacteria such as staphylococci which were no
longer sensitive to penicillin started being noticed. This marked the
beginning of the error of antimicrobial resistance.
Increasing prevalence of resistance has been reported in many
pathogens over the years in different regions of the world including
developing countries(Byarugaba, 2005). As MRSA, Pseudomonas
aeruginosa.

5. Overview
Throughout history, there has been a continual battle between
humans and the multitude of microorganisms that cause infection and
disease.
Examples:
Bubonic plague, tuberculosis, malaria, and more recently, the human
immunodeficiency virus/acquired immunodeficiency syndrome
pandemic, have affected substantial portions of the human population,
causing significant morbidity and mortality.

6. Definitions
Antimicrobial resistance (AMR):
• 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
•sothat 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)

7. (cont.)
Multi-drug resistance (MDR)
• Is defined as having acquired non-susceptibility to at least one
agent in three or more antimicrobial categories.
Extensive drug resistance (EDR)
• Is defined as non-susceptibility to at least one agent in all but
two or fewer antimicrobial categories (i.e. bacterial isolates
remain susceptible to only one or two categories).
Pandrug-resistant (PDR)
• Is defined as non-susceptibility to all agents in all antimicrobial
categories.

8. 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 ).

9. 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 resistance

10. (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.

11. MECHANISMNATURAL RESISTANCE
AGAINST
ORGANISM
Lack of oxidative metabolism
to drive uptake of
aminoglycosides
AminoglycosideAnaerobic bacteria
Inability to reduce drug to
active form
MetronidazoleAerobic bacteria
Lack of PBPsAztreonamGram-positive bacteria
Lack of uptake(increase
thickness of PG layer)
VancomycinGram-negative bacteria
Beta-lactamaseAmpicillinKlebsiella spp.
Beta-lactamaseImipenemStenotrophomonas.
maltophila
Lack of appropriate cell wall
precursor target
VancomycinLactobacilli and Leuconostoc
Lack of uptake resultingSulfonamides, trimethoprim,
tetracycline, or
chloramphenicol
Pseudomonas aeruginosa
Lack of sufficient oxidative
metabolism to drive uptake of
aminoglycosides
AminoglycosidesEnterococci
Lack of PBPsAll cephalosporins

12. (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
physical or chemical agents that alter base sequences.

13. (cont.)
Other types of mutation:
1. Point mutation → change in single base-pair in the DNA.
2. Substitution → replacement of an original base-pair or sequence of
base-pair by another, may be transition (same) or transversion
(different).
3. Deletion.
4. Insertion.
5. Silent.
6. Reading frame shift mutation.
7. Non-sense.
8. Missense.
9. Lethal mutation.
10. Back mutation.
11. Condition lethal mutation.
12. Suppressor mutation.

15. (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.

16. (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
 Transposon contains two inverted repeat, in which the two enzymes are identifying.
 Mobile genetic element are probably responsible for most of the genetic variability in natural
bacterial population, and the spread of bacterial resistance genes.
 Some transposons may contain a special, more complex DNA fragment called ‘‘integron’’, a site
capable of integrating different antibiotic resistance genes and thus able to confer multiple
antibiotic resistance to a bacteria. Integrons have been identified in both gram-negative and
gram-positive bacteria, and they seem to confer high-level multiple drug resistance to the
bacteria that carry and express them

21. MECHANISM INVOLVEDRESISTANCE OBSERVEDACQUIRED RESISTANCE
THROUGH
Point mutations in the
rifampin-binding region of
rpoB
Mycobacterium tuberculosis
resistance to rifamycins
Mutations
Mutations in the
chromosomal gene specifying
dihydrofolate reductase
E.coli, Hemophilius influenzae
resistance to trimethoprim
Via acquisition of mecA genes
which is on a mobile genetic
element called “staphylococcal
cassette chromosome”
(SCCmec) which codes for
penicllin binding proteins
(PBPs) that are not sensitive to
ß-lactam inhibition
Staphylococcus aureus
resistance to methicillin
(MRSA)
Horizontal gene transfer

22. Major biological mechanisms of antimicrobial
resistance
 Whichever way a gene is transferred to a bacterium, the development of
antibiotic resistance occurs when the gene is able to express itself and produce a
tangible biological effect resulting in the loss of activity of the antibiotic.
 Microbes utilize numerous mechanisms of resistance to antimicrobial
Drugs they can be summarized as follow:
I. Decreased uptake and increased efflux of drug from the microbial
cell.
II. Expression of resistance genes that code for an altered version of the
substrate to which the antimicrobial agent binds.
III. Covalent modification of the antimicrobial drug molecule which
inactivates its antimicrobial activity.
IV. Increased production of a competitive inhibitor of antibiotic.
V. Drug tolerance of metabolically inactive persisters.
VI. Biofilms.
VII. Swarming.

23. (cont.)
I. Decreased uptake(impermeability) and
increased efflux of drug from the microbial cell.
• Decreased uptake of antimicrobial drugs and/or use of transmembrane
efflux pumps prevents the concentration of antimicrobial agent from
increasing to toxic levels within the microbial cell (↓uptake↓conc↓effect).
• Gram negative bacteria have an outer membrane surrounding a periplasmic
space (which contains a peptidoglycan cell wall),which surrounds an
innermembrane, whereas Gram positive bacteria have a peptidoglycan cell
wall surrounding only a single plasma membrane.
• This outer membrane may provide an extra barrier against
drug uptake (especially hydrophobic drugs) in Gram
negative bacteria, which is not present in Gram positive
bacteria. This is one explanation why Gram negative
bacteria are less susceptible than Gram positive bacteria to
many antibiotics, including beta-lactams and macrolides.

24. (cont.)
• E.g. P. aeruginosa and E.coli are containing proton-dependant
efflux pump which expel the drug outside the cell.
• Exampls
Tetracyclin resistance byTetA,B and k gen mediated efflux pump.
Fluroquinolon resistance by decreas uptak
Vancomycin resistance By increas thickness of bacterial cell wall,
so decreas uptak.
 EFFLUX
 AND
 IMMPERMEABILITY

25. (cont.)
II. Expression of resistance genes that code for an altered
version of the substrate to which the antimicrobial agent
binds
 GENE mutation → translated to altered protein( substrate) → low binding
affinity→ reduced antibacterial activity → resistance developed.
 E.g.
• MacA resistance gene codding for PBP2A (altered form
than wild-type), represent resistance of MRSA against B-
lactams.
• VanA resistance gene codding for altered binding
substrate (D-alanine–D-lactate ligase, Vancomycin has
1000 times lower affinity for D-alanine–D-lactate than D-
alanine–D-alanine, so the VanA gene confers resistance
to vancomycin. Both vancomycin resistant Enterococcus
(VRE) and vancomycin-resistant S. aureus (VRSA) express
VanA.

26. (cont.)
• Expression of altered DIHYDROFOLATE PETROATE represent sulfonamide
resistance, Bacteria using this resistance mechanism include S.
pneumoniae, S. pyogenes, Neisseria meningitidis, and E. coli.
• Altered gyrA and gyrB, represent resistance of Gm-ve against Quinolones.
• Altered Topoisomerase IV, represent resistance of Gm+ve against
Quinolones.

27. (cont.)
III. Covalent modification of the antimicrobial drug
molecule which inactivates its antimicrobial activity.
 Microbes can also express drug resistance genes that code for
enzymes that covalently modify the antimicrobial drug, thereby
reducing its antimicrobial activity.
 E.g.
i. beta-lactamases hydrolyze the beta-ring of betalactams,
thereby inactivating the antibiotic activity of the beta-
lactam molecule and conferring beta-lactam resistance.
ii. ACT N-acetyltransferse, which acetylates an NH2 group of
the aminoglycoside molecule.
iii. APH O-phosphotransferase, which phosphorylatesan OH
group of the aminoglycoside molecule.
iv. and the ANT O-adenyltransferase, which adenylates an OH
group of the aminoglycoside molecule.
v. Acetyltransferases, which acetylate and thereby inactivate
chloramphenicol.

29. (cont.)
IV. Increase production of competitive inhibitors.
 Bacteria can also achieve antibiotic resistance by
synthesizing a molecule that is a competitive inhibitor
of the antibiotic(Enzyme Substrate).
Example
 Mechanism of sulfonamide resistance is increased
synthesis by bacteria of para-aminobenzoic acid
(PABA), which competes with the sulfonamide drug
for the binding site of bacterial dihydropteroate
synthetase.
 This mechanism of sulfonamide resistance is used by S.
aureus and N. meningitidis.

30. (cont.)
V. Drug tolerance of metabolically inactive persisters.
 The presence of metabolically inactive persisters at the site
of infection in close to actively bacterial population, results
in antibacterial tolerance.
 Recurrence of infection after treatment is usually occur.
 This mechanism occur due to expression of gene called
Toxin-Antitoxin, which cause their metabolic activity to
slow or stop.
 After the hos exposed to antibacterial agent, the actively
metabolic bacterial of population eradicated.
 And the persisters are turn to metabolically active and
cause recurrence of infection.

31. (cont.)
VI. Biofilm.
Biofilm formation can result in tolerance of bacteria to very high
concentrations of multiple antibiotics, resulting in chronic
infections despite antibiotic treatment.
 Steps of biofilm
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.

32. Biofilm steps

33. Role of Extracellular polymeric substance in resistanse
I. Act as barrier to diffusion of oxygen and nutrients. In turn the
deeply located bacteria to metabolically in active and tolerate
antibacterial agent rather than superficially located bacteria.
I. 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
 Third, enzymes within the EPS matrix also covalently modify
antibiotic molecules, thereby inactivating their antimicrobial
activity.

34. (cont.)
VII.Swarming.
 type of multicellularity in bacteria and operates by the
following mechanism:
I. Planktonic bacterial cells differentiate into elongated
cells with multiple flagella (swarm cell).
II. More swarm cell adhere together and act as single unit.
These swarm cells are also tolerant to antimicrobial
agent.
III. Subculturing of swarm cell in a liquid media, reverse
back to planktonic bacteria which no longer have
tolerance to antibacterial agent.
 E.g. Bacillus subtilis, Serratia marcescens, E. coli, Salmonella
typhimurium and P. aeruginosa.
 Planktonic form: are single-cells that may float or swim in a liquid
medium.

35. Factors that promote bacterial resistance
suboptimal use of antimicrobials for prophylaxis and
treatment of infection.
 noncompliance with infection-control practices.
 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
 grouping of colonized patients in long-term-care facilities.
 antibiotic use in agriculture and household chores.
 increasing national and international travel.
Lack of education and poverty.

36. Consequence of antibacterial resistance

37. Antibacterial in food and animal industries
 Veterinary antibiotics (VAs) are widely used in many countries worldwide to treat disease
and protect the health of animals.
 They are also incorporated into animal feed to improve growth rate and feed efficiency.
 As antibiotics are poorly adsorbed in the gut of the animals, is excreted unchanged in faeces
and urine. Given that land application of animal waste as a supplement to fertilizer.
 there is a growing international concern about the potential impact of antibiotic residues on
the environment.
 E.g. tetracycline, chloramphenicol, triclosan and bacitracin.

39. 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.

40. 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.
(2004)
ErythromycinRosmarinus officinalisCarnosic acid carnosol
Shibata et al. (2005)B-lactamsCaesalpinia spinosaEthyl gallate
Gibbons et al. (2004)
Hu et al. (2002)
Zhao et al. (2001)
Norfloxacin
Imipenem
Panipenem
B-Lactams
Camellia sinensisEpicatechin gallate
Epigallocatechin
gallate

41. 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.

43. Finally

44. So………..