Description of the major classes of antimicrobial drug, resistant mechanisms developed by bacteria to combat the action of antimicrobials, and the control measures needed to limit this horizontal gene transfer.

1. Multi-Drug Resistant
Organisms
(MDROs)
Dr Mostafa Mahmoud, MD, Ph D,
Consultant Microbiologist
Assist. Prof. of Medical Microbiology &
Immunology

2. Genetic materials controlling bacterial
functions are:
• 1- Chromosome
• 2- Mobile genetic elements (plasmids, transposons, genetic
islands).

3. Bacterial cell structure (Prokaryote)

4. Classification and mechanism of action of antimicrobials
• Antimicrobial agents are substances that kill or inhibit the growth
of microorganisms and are suitable for systemic use.
• If this antimicrobial substance is synthesized in the laboratory it is
named Chemotherapeutic.
• The term “antibiotic” is a substance produced as a secondary
metabolite by a bacterium which inhibits or kills other
microorganisms.

5. Some Naturally Produced Antibiotics
Species Microorganism Antibiotic produced
Gram positive Bacteria Bacillus subtilis Bacitracin
Bacillus polymixa Polymixin
Actinomycetes Micromonospora purpurea Gentamycin
Streptomyces Streptomyces erythreus Erythromycin
Streptomyces griseus Streptomycin
Streptomyces rimosus Tetracycline
Streptomyces orientalis Vancomycin
Fungi Penicillium chrysogenum Penicillin
Cephalosporium acremonium Cephalosporins
Miscellaneous Pseudomonas fluorescens pseudomonic acids*

6. classifications of antimicrobials:
• Several ways according to:
• Mechanism of action: (inhibit cell wall, protein, or NA synthesis
etc).
• Spectrum of activity: (broad or narrow spectrum).
• Killing or inhibitory effect upon microorganism: (Bacteriostatic or
Bactericidal).
• The chemical structure.

7. The classification of AB by the mechanism of action

8. 1- Inhibition of bacterial cell wall synthesis
Group Examples Spectrum of action
Penicllins: 1-Natural Penicillin G (injection)
Penicillin V (oral)
Gram positive bacteria.
2-Penicillinase-Resistant
Penicillins
Cloxacillin - Dicloxacillin - Methicillin,
Nafcillin - Oxacillin
Antistaphylococcal actions
3- Aminopenicillins Amoxicillin - Ampicillin
Amoxicillin/clavulanate
Ampicllin/sulbactam
bacapicillin
Gram +ve and Gram -ve
bacteria.
4- Carboxypenicillins Carbenicillin - Ticracillin,
Ticracillin/clavulante
Greater activity against gram
negative organisms
5-Ureidopenicillins and
Piperazine
Mezlocillin - Piperacillin
Piperacillin/tazobactam
They have the broadest-
spectrum of all penicillins
especially on Pseudomonas
aeruginosa

9. Group Examples Spectrum of action
2-
Cephalosporins:
1st generation
Cefadroxil - Cefazolin - Cephalexin
Cephalothin - Cephradine
Gram (+ve) & few Gram (–ve) e.g.
E. coli, Klebsiella
2nd generation Cefaclor – Cefamandole - Cefmetazole,
Cefoxitin - Cefoprzil - Cefuroxime
More G (–ve) e.g. Klebsiella,
Proteus, less on (G+ve)
3rd generation Cefixime – Cefoprazone - Cefotaxime,
Ceftazidime - Ceftriaxone
Pseudomonas & Enterobacter.
- TTT of HAIs.
4th generation Cefepime upon G (+ve) & G (-ve)
organisms, including
P. aeruginosa

10. Other cell wall inhibitors:
Group Examples Spectrum of action
3- Carbapenems Imipenem - Meropenem
Etrapenem - Doripenem
broad-spectrum of activity
(MSSA- Pseudomonas).
4- Glycopeptides Vancomycin
Bacitracin
Teicoplanin
MRSA
5- Monobactams Aztreonam Aerobic Gram-negative
microorganisms
N.B. - Beta-Lactam Antimicrobials include: PENICLLINS, CEPHALOSPORINS,
CARBAPENEMS, MONOBACTAMS.
- All Beta-Lactam Antimicrobials are generally Bactericidal on bacteria.

11. 2- Interference with cell membrane function
• This group includes some antibacterial agents e.g. polymyxin B
and colistin, and antifungal agents e.g. amphotricin B, imidazoles
and nystatin.

12. 3-Inhibition of bacterial protein synthesis
Group Examples Mechanism of action Effect bacteria
Aminoglycosides Streptomycin - Neomycin,
Kanamycin - Tobramycin,
Netilimicin - Amkacin
Irreversible binding to 30S
ribosomal subunit
Bactericidal
Tetracyclines Tetracycline - Oxytetracycline
Demeclocycline – Doxycycline
Minocycline
Reversibly bind to 30S
subunit
Bacteriostatic
(Chlamydia &
rickettsia)
Glycocyclines
(30S= TAGG)
Tigecycline Binds to 30S ribosomal
subunit.
Bacteriostatic
(mostly)
(for MRSA, GISA,
ESBL, and VRE)

13. Group Examples Mechanism of action Effect bacteria
Chloramphenicol Chloramphenicol Binds 50S and inhibits
peptidyl transferase
Bacteriostatic
Clindamycin Clindamycin Binds to 50S Bacteriostatic
(Anaerobes)
Macrolides Erythromycin, azithromycin,
clarithromycin Dirthromycin,
Troleandomycin
Bind to the 50S subunit
inhibiting RNA-
dependent protein
synthesis.
Bacteriostatic or
Bactericidal
Ketolides Telithromycin Binds to the 50S subunit. Bacteriostatic or
Bactericidal
Inhibitors of protein synthesis (continue

14. Group Examples Mechanism of action Effect bacteria
Oxazolinones Linezolid Binds to the 50S subunit Bacteriostatic or
Bactericidal
(for VRE, MSSA and
MRSA)
Streptagramins Quinupristin-dalfopristin
(Synercid)
binding to the 50S
ribosomal subunit of
gram + bacteria
Bactericidal
Cyclic lipopeptides Daptomycin Not completely
understood, but alters
cell membrane activity
Bactericidal
Inhibitors of protein synthesis (continue)

15. 4- Inhibition of nucleic acid synthesis
Group Examples Mechanism of
action
Effect on bacteria
Fluoroquinolone (1st) Nalidixic acid
(2nd)–Ciprofloxacin - Levofloxacin
Norfloxacin - Ofloxacin
(3rd)–Gatifloxacin
(4th)–Trovafloxacin
Inhibit DNA gyrase Bactericidal
Sulfonamides Sulfisoxazole - Sulfamethoxazole,
Sulfadiazine - Sulfadoxine,
Sulfasalazine - Sulfapyridine
Inhibit folic acid
(FA) synthesis.
Bacteriostatic

16. Group Examples Mechanism of action Effect on bacteria
Trimethoprim Trimethoprim Inhibit dihydrofolate reductase
enzyme
Bacteriostatic
Bactericidal if combined
with sulfa
Cyclic
lipopeptides
Daptomycin Not clearly understood,
disruption of DNA, RNA and
protein synthesis
Bactericidal
Natural
For MRSA & VRE
Rifamycins Rifampin,
Rifabutin,
Rifapentine.
Inhibiting RNA by binding to
DNA-dependent
RNA polymerase
Bactericidal
(Antibiotics)
TTT of TB
Inhibitors of nucleic acid synthesis (continue).

17. Mechanisms of antimicrobial drug resistance
Classification:
• The resistance of microorganisms to antimicrobials is classified
as being either natural or acquired.
• 2.3.2.1 Natural resistance
• An organism is termed as having natural (intrinsic) resistance
when it has an inherent resistance to the action of an
antibiotic; this pattern of resistance is common to all isolates
of the species, e.g. the resistance of Escherichia coli to
macrolides and the resistance of Pseudomonas aeruginosa to
most drugs.

18. • The intrinsic (natural) resistance of a microorganism to an
antimicrobial is explained by the absence or inaccessibility of
the target of the drug action, e.g. Gram-negative bacteria are
naturally resistant to some antibiotics e.g. erythromycin due to
the non-permeability of the outer membrane

19. Acquired resistance
• Acquired resistance is developed to an antibiotic to which the
microorganism was previously susceptible; it develops within one
or more isolates of the species, i.e. not all strains of a species are
resistant.
• For an antimicrobial to produce its intended action, it has to have
a target for its action (e.g. in the form of an enzyme or protein)
within the bacterial cell, to be able to reach this target, and also
to reach the target in its active form, i.e. not having been
destroyed.
The functional mechanisms of acquired resistance

20. What bacteria can do to combat antimicrobials?
• 1) they may destroy or inactivate the antibiotic;
• 2) bacteria can use an efflux system to exclude the drug from its
interior ;
• 3) bacteria can produce alterations in the target site used by
antimicrobials to act or they may completely prevent this binding;
• 4) bacteria can reduce their cell surface permeability or even
completely block the entrance of the antimicrobial to the cell, so
that the antimicrobial can no longer act; and
• 5) bacteria can produce a bypass mechanism by using alternative
pathways which are different from those inhibited by the antibiotic

21. Different mechanisms of antimicrobial resistance
used by bacteria
Mechanism Examples of affected antimicrobials
1- Destruction, modification, or
inactivation of the antimicrobial.
- β-lactam antibiotics - Chloramphenicol
- Aminoglycosides
2- Multidrug efflux pumps. -Tetracycline
3- Target site alteration. -β-lactam antibiotics - Chloramphenicol
- Streptomycin - Quinolones
- Fusidic acid - Erythromycin
– Glycopeptides - Rifampicin

22. Different mechanisms of antimicrobial resistance
used by bacteria
Mechanism Examples of affected antimicrobials
4- Reduction in the cell surface
permeability or access of the
antimicrobial to the cell interior.
Tetracyclines - Quinolones
β-lactam antibiotics
Aminoglycosides - Chloramphenicol
5- New metabolic bypass
mechanism.
Trimethoprim - Sulphonamides

23. The basis (mechanism) of antimicrobial resistance
• A-Passive (intrinsic or phenotypic) drug resistance:
• bacteria stop multiplying they are not affected by the
antimicrobial; Mycobacterium tuberculosis, where “persister”
• L-form bacteria, which lack the cell wall which is the target for
action by some antimicrobials like penicillins and cephalosporins;
this is sometimes termed “phenotypic tolerance” rather than
resistance

24. B-Active drug resistance:
• Attributed to the emergence or acquisition of a new gene(s) which
controls the process of resistance either following mutation, which
may be spontaneous or induced, or by the transfer of a gene from
another bacterium or another locus within the same bacterium by
the process of transposition

25. 1- Mutation
• Mutation is a heritable change in the structure of the genes which
may arise spontaneously as an error of replication.
• Due to UV light, radiation, or alkylating agents
• Termed chromosomal resistance, as it usually originates in a
chromosome as a spontaneous mutation in a locus responsible for
the antimicrobial drug action.
• May arise by insertion or deletion of nucleotide(s).

26. Examples of Mutations:
1-Mutation producing a single amino acid change in the PBPs
gives low level resistance to penicillins and cephalosporins.
2-Mutations in the 23S ribosomal RNA gene also lead to
linezolid-resistant strains of VRE and MRSA,

27. Mobile Genetic Elements (carriers of antimicrobial
resistance genes (ARGs)) from one species to another??)
• Spread of antimicrobial resistance genes (ARGs) among, human,
animal, and environmental bacteria is mediated through this
mobile genetic elements (MGEs).
• The MGEs include: conjugative plasmids, gene cassettes within
the integrons, plasmids, transposons, and Insertion Sequence (IS)
elements
2- Gene Transfer

28. 1- Conjugative plasmids
• Conjugative plasmids are those having the ability to transfer
themselves and other plasmids from one bacterial cell to
another carrying ARGs present in both Gram positive and Gram
negative bacteria.
• Spread of conjugative plasmids can occur at narrow or wide
range of species.

29. 2- Integrons
• Integrons, which are present naturally as gene expression elements
as they contain open reading frames (ORFs) enabling them to
express the genes it contains, are formed from two conserved
flanking regions which incorporate one or more resistance gene(s)
in-between.
3-Gene cassettes
• The mobile genetic elements (MGEs) within the integrons are
known as the genetic cassettes. Many identified genetic cassettes
are known and have been identified that mediate resistance to
several antimicrobials e.g. penicillins, cephalosporins,
aminoglycosides, chloramphenicol, and trimethoprim.

30. 4- Insertion sequences and transposons
• The simplest transposable DNA sequences are known as the
insertion sequence (IS) elements. They are a heterogeneous class
of MGEs in bacteria, having the ability to promote their own
translocation and do not carry antimicrobial resistance genes
(ARGs).
• Transposons are mobile genetic elements which contain self-
transmissible elements, including transposase and recombination
DNA segments (e.g. ARGs).

32. Slide comment for the figure:
• Transposable DNA elements: a) the insertion sequence (IS) elements which are
the simplest transposable DNA sequences, having reversed identical sequences
(inverted repeats: IR) of 10-40 nucleotides at both ends flanking the
transposase (tnp) gene. The direct repeats formed from 5-9 bp at the
extremities of the structure are the target for the enzyme transposase during
the integration process; b) the simple Tn3 transposons containing the
transposase gene tnpA, regulator sequence (tnpR) and the (res) site to which
the resolvase enzyme binds; c) the composite transposons which are formed
from two IS elements making a frame for a region which is not essential for
transposition e.g. tetracycline resistance gene (tetB); d) represents the
conjugative transposons which have certain segments encoding factors used in
the control of the transfer (Tra) and transposition (Tn) processes

33. Horizontal (lateral) gene transfer (HGT)
• Gene transfer between different bacteria occurs through one of
three mechanisms: conjugation, transformation, or transduction.
Conjugation is the most frequent mechanism mediated in HGT.
1- Conjugation
• Conjugation is the process of gene transfer between bacteria
from the donor to the recipient via intimate contact, termed
“mating through a channel”.
• The transposable DNA elements include the insertion sequences
(IS) and the different forms of tranposons (simple, composite,
and conjugative transposons).

34. Transfer of a conjugative plasmid by the process of conjugation between two bacterial
cells via the sex pili: a) formation of the conjugation channel, b) start of the transfer of
a single strand of the plasmid cleaved by the endonuclease enzyme at a specific point,
c) the cleaved strand entering the recipient cell where d) a complementary strand is
synthesized

35. 2- Transformation
• Bacteria can take exogenous DNA (genes) from the surrounding
environment. This ability to take exogenous genes is called competence
and it is encoded by chromosomal genes within the bacterium, and it is
mostly a time-limited process and occurs in a wide variety of bacteria
e.g. Hemophilus, Helicobacter, Campylobacter, Niesseria, Staphylococci,
and Pseudomonas species.
• Transformation can occur as natural process or can by induced by
certain factors e.g. nutrient access, altered growth conditions, or
starvation.
• Antimicrobial resistant gene (ARG) transfer is the natural one which can
transmit resistance gene among different bacterial strains. The DNA is
up taken as double stranded one which then converted to one strand
while passing the inner membrane.

36. 3- Transduction
• In transduction, the bacterial genes are transferred between
different bacterial strains by the means of bacteriophage, which is
a bacteria infecting virus.
• The phage for transfer must be the temperate (Lysogenic) one and
not the lytic phage.
N.B. Transposition
• In transposition, a DNA segment (mobile genetic element) can
move either to another locus in the same molecule (chromosome
or plasmid) or transfer between them i.e. inside the cell not
involved in HGT.

37. Schematic representation of different gene transfer mechanisms in bacteria: A= transformation, B= Transduction, and C=
Conjugation

38. Source of Resistant Bacteria
• Large amounts of antibiotics are used for human therapy, as well
as for farm animals and even for fish in aquaculture, resulted in
the selection of bacteria resistant to multiple drugs.
• These environmental resistant bacteria carry great harm to human
beings when genes are transferred to clinical isolates by HGT.
• MDROs are those resistant to more than 2 classes of
antimicrobials!!.
• Examples of MDROs include: MRSA, ESBLs, VRE,
• Pan-Resistant organisms are isolated nowadays resistant to all
available antimicrobials.
• Going to the pre-antibiotic era is the actual threat.

39. Various routes
for antimicrobial
resistance gene
spread from
human activity
origins to the
environment

40. How complex the
spread of ARGs
In the community
& Environment

41. Example of
MDROs
Resistant to: Treatment
MRSA Penicillins, Cephalosporins, Monobactams,
Carbapenems. (additionally to aminoglycosides,
macrolides, tetracycline, chloramphenicol, and
lincosamides).
Vancomycin,
Linezolid,
ESBLs Penicillins, cephalsporins, Monobactams. Synercid,
Tigecyclines,
VRE, VRSA
& VISA
Penicillins, Cephalosporins, Monobactams,
Carbapenems, Vancomycin.
Linezolid
PDROs All available antimicrobials Colistin ??
CRE imipenem, meropenem, doripenem, or ertapenem
(Escherichia coli, Klebsiella oxytoca, Klebsiella
pneumoniae, or Enterobacter)

42. CDC definitions of MDROs
• MRSA: Includes S. aureus cultured from any specimen that tests
oxacillin-resistant, cefoxitin-resistant, or methicillin-resistant by
standard susceptibility testing methods (AST) OR, positive FDA
approved direct testing from sampling (e.g. PCR) for MecA gene.
• VRE: Enterococcus faecalis, Enterococcus faecium, or Enterococcus
species unspecified that is resistant to vancomycin, by standard
susceptibility testing methods (AST) or by direct testing by FDA
approved test e.g. PCR for genes (VanA, VanB, VanC).

44. • CRE (Carbapenem-Resistant Enterobacteria): Any Escherichia
coli, Klebsiella oxytoca, Klebsiella pneumoniae, or Enterobacter
spp. testing resistant to imipenem, meropenem, doripenem, or
ertapenem by standard susceptibility testing methods (AST). OR
by production of a carbapenemase (i.e., KPC, NDM, VIM, IMP,
OXA-48) genes by PCR.

45. • MDR-Acinetobacter: Any Acinetobacter spp. testing non-
susceptible (i.e., resistant or intermediate) to at least one
agent in at least 3 antimicrobial classes of the following 6
antimicrobial classes:
Antimicrobial
class
β-lactam/β-lactam β-
lactamase inhibitor
combination
Aminoglycosides Carbapenems
Representatives Piperacillin
Piperacillin/tazobactam
Amikacin
Gentamicin
Tobramycin
Imipenem
Meropenem
Doripenem
Antimicrobial
class
Fluoroquinolones Cephalosporins Sulbactam
Representatives Ciprofloxacin
Levofloxacin
Cefepime
Ceftazidime
Ampicillin/sulbact
am

46. Burdens of MDROs Problem
1- Antimicrobial resistance kills (no treatment).
2- Antimicrobial resistance hampers the control of infectious
diseases.
3- Antimicrobial resistance increases the costs of health care.
4- Antimicrobial resistance jeopardizes health care gains to
society.

47. MDRO Prevention and Control
1. Administrative support: (Financial and HR, communication
system, HH facilities, staff levels, adherence to IPC
recommendations).
2. Education: Facility-wide, unit-targeted, and informal, educational
interventions.
3. Judicious use of antimicrobial agents: (antimicrobial stewardship
program). Use narrow spectrum, treat only infections not
contaminant or colonizers, duration limited, restricted Abs
validation).

48. Control of MDROs Spread:
4. MDRO surveillance: (new pathogen, trends, effective
interventions, by either reviewing micro lab results or by Active
Surveillance Culture/Testing (ASC/AST) to detect colonization.
• Antibiograms (simplest for of MDROs surveillance).
• MDRO Infection Rates reviews
• Molecular typing of MDRO isolates.
• Surveillance for MDROs by Detecting Asymptomatic Colonization
(great impact up to 65% reduction of spread ).

49. Methods for obtaining ASC/AST:
• MRSA: Studies suggest that cultures of the nares.
• VRE: Stool, rectal, or perirectal swabs.
• MDR-GNBs: peri-rectal or rectal swabs + oro-pharyngeal,
endotracheal, inguinal, or wound cultures.
• Rapid detection methods: (media containing chromogenic enzyme
substrates – real-time PCR-based tests for MRSA from swabs, vanA
and vanB genes (VRE or VRSA) from rectal swabs).

50. 5. Infection Control Precautions: (Standard and Contact isolation
Precautions for MDROs, Hand hygiene) .
• Cohorting and other MDRO control strategies: (cohorting of
patients, cohorting of staff, use of designated beds or units, unit
closure are necessary for control of transmission.
• Duration of Contact Precautions: controversial. 3 negative swabs,
better for all period of stay in facility.
• Barriers used for contact with patients infected or colonized with
MDROs: (gloves with or without gowns, .
• Impact of Contact Precautions on patient care and well-being:
(adverse effects).

51. 6. Environmental measures e.g. surfaces and medical equipment,
environmental cultures are not recommended routinely, .
• Stick to proper environmental , surfaces, and equipment
cleaning).
7. Decolonization: (treat colonized persons with MRDOs to
eradicate them e.g. MRSA, little success in VRE).

52. Stop the Abuse of Antimicrobials
Follow antimicrobial Policy and
stewardship program in
hospitals.

53. Stop the misuse of antibiotics by the community

56. Control of antibiotic usage for non-therapeutic
purposes

59. If the world does not cooperate
together fighting microbial
resistance, then all these drugs
will have no value.
Eventually we will go to the pre-
antibiotic era.

60. References:
• http://www.who.int/mediacentre/factsheets/fs194/en/.
• http://www.cdc.gov/hicpac/mdro/mdro_4.html.
• GCC Infection Control Manual 2013.

61. Thank You