MEDICAL MICROBIOLOGY

Course for medical students

Associate Professor L. Yocheva, Ph.D.

Department of Biology, Medical Genetics and

Microbiology
Medical Faculty
SU “St. Kliment Ohridski”

Email: [email protected]
 Lecture 7
Antimicrobial Chemotherapy
(Part one)

1. Antimicrobials - definition, principles of

action, requirements, spectrum of activity,
names, side effects.
2. Major groups of antibiotics. Mechanism of
action, main representatives.
3. Inhibitors of the cell wall synthesis.
 Key words

• Antibiotics
• Chemotherapeutic agents
• Selective toxicity
• Mechanism of action
• Spectrum of activity
• Side effects
• Combined therapy
• Beta-lactams
• Glycopeptides
 Anti-Infective Agents

• Preparations directed against

infectious agents

• Antibacterial, antiviral, antifungal and

antiprotozoal drugs

• Chemotherapeutics are synthetic

chemicals

• Antibiotics are mold or bacteria-derived

products
 Antibacterial Agents or Antimicrobials
• Most antibacterial drugs are antibiotics
• Types of antibiotics:
• Natural antibiotics (bacterial or mold products)
• Semisynthetic (modified mold or bacterial products)
• Synthetic (chemically synthesized biological
products)

• Some chemotherapeutics posses antibacterial

activity

• Antibiotics and some chemotherapeutic agents are

called antimicrobial agents or antimicrobials

• However, the term antibiotic is often used for all

types of antimicrobial agents
 History of Antimicrobial Chemotherapy
• 1910, Paul Ehrlich
• Introduced the arsenic-based
drug Salvarsan as a remedy for
syphilis
• The discovery of a specific drug
to treat a specific disease marked
the beginning of antimicrobial
chemotherapy

Paul Ehrlich
1854-1915

Salvarsan treatment kit

for syphilis, Germany, 1909–1912
 History of Antimicrobial Chemotherapy
• 1929, Alexander Fleming
• Discovered the penicillin
• He concluded that some molds produce
a substance, that can kill bacteria
• He published his observation in British
Journal of Experimental Pathology
• 1945, Nobel Prize
1881–1955

Penicillium notatum
 History of Antimicrobial Chemotherapy
• 1935, Gerhard Domagk
• Discovered Prontosil - the first
commercially available
sulfonamide (active against
Gram-positive bacteria)
• 1939, Nobel Prize in Physiology
or Medicine

Gerhard Domagk
1895–1964

Tube of Prontosil tablets,

Germany, 1935-1950
 History of Antimicrobial Chemotherapy
• 1940, Howard Florey, Ernst Chain
and E. Abracham isolate, purify and
concentrate penicillin as a
substance, for which they receive a
Nobel Prize
• 1943 – Penicillin was introduced
as a drug. Мass production of
penicillin was followed
H. Florey
• 1942, Selman Waksman
discovered the
second antibiotic-
Streptomycin
E. Chain

S. Waksman
 General Criteria to be Met for Antibiotics

• To be selectively toxic – the most important

characteristic
• To be soluble and stable in the
macroorganism
• To reach the infected tissue
• To penetrate well into human cells (in the case
of intracellular infections)
• Do not accumulate
• Do not cause an allergic reactions
• Do not induce antibacterial resistance
• Do not suppress the normal microbial flora
 Selective Toxicity

• Ability of antibiotics to damage only the

microorganism, not the macroorganism

• Selective toxicity is sometimes also

determined by the dose of the antibiotic

• Disinfectants and antiseptics are not

selectively toxic
 Selective Toxicity

• Differences in the structure of prokaryotic

and eukaryotic cells determines the ability
of antibiotics to act as selective toxic agents
against bacterial cells
 Characteristics of the Selective
Toxicity

• Selective toxicity is realized by

different approaches:
– Unique targets must be present in the
pathogen and absent in the host
– The target must be structurally different
in the pathogen vs. the host
 Antibiotics - Mechanisms of Action

• The antibiotics can inhibit synthesis or

function of all essential bacterial
structures:
– cell wall
– cell membrane
– ribosomes
– DNA
 Antibiotics - Mechanisms of Action

Beta-
lactams

Aminoglycosides
 Antibacterial Spectrum

• Narrow spectrum: against a limited

number of bacteria (preferentially active
against either Gram-positive or Gram-
negative bacteria)

• Broad-spectrum: against a variety of

bacteria (Gram-positive, Gram-negative;
also intracellular bacteria, anaerobes,
etc).
 Clinical Use of Antibiotics for Treatment
• N.B. Antibiotic treatment should be initiated only when
it is necessary
1. Etiologic therapy – when the causative agent of a
infection is known (confirmed in a laboratory with
defined antibiotic susceptibility)
2. Empiric therapy - when the causative agent is
unknown:
˗ In life threatening infections: start with empirically
chosen broad-spectrum antibiotics; once the
organism is identified and its antimicrobial
susceptibility determined, a more appropriate
specific therapy should replace the “umbrella”
therapy
˗ Based on experience in infections that a not very
severe
Clinical Use of Antibiotics for Prophylaxis

• In some cases antibiotics are used for

prophylaxis:
– Surgery (e.g. patient undergoing lower
bowel resection receive perioperative
antibiotic treatment)
– Congenital abnormalities
– Neutropenic patients (neutropenia: when the
PMN cells fall below a lower limit of 1500
cells per µl (1.5x109 cells/l)
– Healthy persons that have a contact with
dangerous infectious diseases
 Antimicrobial Drugs Used in Combination

• To treat polymicrobial (mixed) infections;

common in brain abscesses, pelvic
infections, and infections from perforation of
abdominal organs

• To delay or block the emergence of microbial

resistance to one drug by the use of a second
or third drug

• To treat very serious microbial infections

• To achieve synergistic effect of antibiotics

 Synergistic Antimicrobial Effect

• The combined action is significantly

greater than the sum of effects
• Examples:
– Affecting the cell membrane or cell wall from
some antibiotics can facilitate the entry of
the second drug
(e.g. penicillin and gentamycin)

– One drug can prevent the inactivation of a

second drug by microbial enzymes
(e.g. beta-lactam antibiotic and beta-
lactamase inhibitor)
Bacteriostatic and Bactericidal Antibiotics
• Bacteriostatic activity – level of antimicrobial
activity that inhibits (suppress) bacterial
growth
• It may depend on the concentration of the drug

• Bactericidal activity – level of antimicrobial

activity that kills the bacteria

• Generally bactericidal drugs kill only growing

bacteria; therefore, concurrent use of
bactericidal and bacteriostatic agents is
usually avoided
 Examples of Bacteriostatic and
Bactericidal Antibiotics
• Bacteriostatic drugs: tetracycline,
sulfonamide, trimethoprim, chloramphenicol,
macrolides, novobiocin, clindamycin

• Bactericidal drugs: beta-lactams, quinolones,

aminoglycosides, polymyxins, rifampin,
bacitracin

• There is no sharp distinction between the two

types of activity, because it depends upon
drug concentration and bacterial species
 Minimum inhibitory concentration
Minimum bactericidal concentration

• Minimum inhibitory concentration – the

lowest antibiotic concentration that inhibits
bacterial growth
• An important factor in determining bacterial
resistance

• Minimum bactericidal concentration - the

lowest antibiotic concentration that kills
bacteria
 Side Effects of Antibiotics

• As a rule, antibiotics are well tolerated from the

patients
• Side effects are possible
• They can affect different systems
• Some antibiotics are restricted for use due to their
toxicity
• Main adverse effects:
˗ Dysbiosis – disruption of the balance of species in
the human microbiome
˗ Toxicity – oto-, nephro-, hepato-, myelo-,
chondrotoxicity; nausea, vomiting, diarrhea
˗ Allergic reactions - urticaria, anaphylactic shock
 Routes of Administration, Dosage and
Duration of Treatment
• Administrated orally, intravenously, intramuscularly;
topical antibiotics are for use on the skin or mucous
membranes
• Benefits and disadvantages of different routes of
application
• Low, medium and high doses
• Short, medium and long-term treatment
 Names of Antibiotics

• Most antibiotics have two names:

• Trade or brand name - created by the drug
company that manufactures the drug
• Generic name - based on the antibiotic's
chemical structure or chemical class

• Trade names are capitalized

• Generics are not capitalized
 Factors of the Host, Influencing the
Effects of Antibiotic Therapy

• Renal or liver function

• Pregnancy
• Age
• Vascularization and drainage of organs and
tissues (impaired blood supply or drainage of
the affected area usually is a problematic for
effective antibacterial treatment)
 Problematic Anatomical Sites and
Diagnoses for Antibacterial Treatment

• It is difficult for antibiotics to reach the bacteria:

– Endocarditis – inflammation of the inner lining of the
heart muscle, (fibrin from inflammation covered the
infectious area)
– Blood-brain barrier and cerebrospinal fluid (CSF)
– Osteomyelitis (bones are sites of relatively poor
vascularity)
– Artificial joints and valves
– Biofilms (covered the bacteria)
– Abscesses (surrounded by capsules)
– Intracellular infections
 Possible Cases of Wrong or Overuse of
Antibiotics

• Prophylactically before surgery

• Empirically (suspected microbial pathogen)
• Unnecessary use of broad spectrum
• Viral infections in children
• Unfinished course or too long course of
treatment
• Improper dosage
• Fever of unknown origin
CELL WALL SYNTHESIS
INHIBITORS
 Antibiotics, that Inhibit Cell Wall
Synthesis

• Group of β-lactam antibacterial agents

• Group of Glycopeptides
 Effect of Activity

• Bactericidal effect

• Act on microorganisms in an active growth

phase
Structure of Peptidoglycan

peptide bridge
 Stages in Peptidoglycan Synthesis.
Antibiotics, that Act at this Levels
Three Stages in Peptidoglycan Synthesis
1. Cytoplasm stage. Disaccharide pentapeptide is
formed. Antibiotics are Fosfomycin and
Cycloserine
2. Membrane stage. Bactoprenol is a transporter of
disaccharide pentapeptide. Antibiotic is Bacitracin
3. Cell wall stage. Disaccharide pentapeptide is
attached to peptidoglycan (transpeptidation). The
pentapeptide is converted to a tetrapeptide.
Antibiotics, that act at this level are Penicillin and
Vancomycin
 Enzymes for Synthesis and Enzymes for
Degradation of Peptidoglycan

• Transpeptidases or carbocypeptidases are

involved in the synthesis of peptidoglycan at
the cell wall level
• They are called penicillin-binding proteins
(PBPs)

• Another enzymes can activate autolysis of

peptidoglycan
 Group of Beta-Lactam Antibiotics

• The most widely used antibiotics

• About 60% of the antimicrobial agents
used in practice are β-lactam antibiotics

• Highly effective with low toxicity

• Lack of toxicity of β-lactam drugs to humans

must be attributed to the absence of cell wall
and peptidoglycan in mammalian cells
 General Characteristics of
β-Lactam Antibiotics

• All have β-lactam ring

• Activity of all classes of beta-lactam

antibiotics depends on intact beta-lactam ring

• PBPs are targets for beta-lactam

antibiotics
 Beta-Lactam Antibiotics
Beta-
lactam ring
 Mechanism of Action of β-Lactam
Antibiotics

1. Binding to PBPs and inhibition of cell wall

synthesis by blocking the transpeptidation
of peptidoglycan
2. Activation of the cell autolytic enzymes

• The final result is blocking the

synthesis of peptidoglycan and lysis
of the cell
 Mechanism of Action of β-Lactam
Antibiotics

• All β-lactam antibiotics have

bactericidal effect
• Act only on the microorganisms in the
active growth phase
• Cell-wall deficient and nongrowing
cells are not killed by beta-lactam
antibiotics
Mechanism of Action of β-Lactam
Antibiotics
 Problems in the Use of β-Lactam
Antibiotics

• A major problem - the development of

bacterial resistance

• Two basic mechanisms:

– Enzymes beta-lactamases inactivate beta-
lactam ring
– Mutations, leading to a change in PBPs
(PBPs lose their ability to bind penicillin)
 Major Groups of β-Lactam Antibiotics

• They are presented in four main

groups:
– Penicillins
– Cephalosporins
– Carbapenems
– Monobactams
 Chemical Structure of Penicillins

• All penicillins are derivatives

of 6-aminopenicilanic acid
• They share the same basic
chemical structure
• Basic structure of Penicillins:
– a thiazolidine ring
– a β-lactam ring
– side chains of the molecule that determine the
pharmacological properties and spectrum of
activity of different kinds of penicillins
 Problems - β-Lactamases and Allergy

• Beta-lactamase: an enzyme that hydrolyzes the

β-lactam ring, thus inactivating the antibiotics
• The enzymes specific for penicillins,
cephalosporins, and carbapenems are
the penicillinases, cephalosporinases,
and carbapenemases
• The resulting product of destruction of β-
lactam ring is penicilloic acid
• It don’t have antibacterial activity, but have an
important role in development of allergy as
being hapten
 Penicillins

• Natural penicillins
• Penicillinase–resistant penicillins -
аntistaphylococcal penicillins
• Broad-spectrum penicillins
˗ Aminopenicillins
˗ Carboxypenicillins
˗ Ureidopenisilins
• Beta-lactamase inhibitors
 Natural Penicillins
• Benzylpenicillins (penicillin G)
• Discovered by A. Fleming and first used on humans
in 1941

• Penicillin G benzathine (depot penicillin)

• Phenoxymethylpenicillin (penicillin V) - for oral use

• Penicillin G and Benzathine penicillin G are

inactivated by gastric acid; thus they are for
parenteral use

• All natural penicillins are susceptible to enzymes

beta-lactamases (penicillinases)
 Spectrum of Activity

• Spectrum – narrow, mostly against Gram-positive

bacteria and most Gram-positive anaerobes:
– S. pyogenes , S. pneumoniae
– Gram-positive anaerobes (Clostridium spp.)
– Gram-positive rods (Corynebacterum spp.)
– Limited activity against staphylococci (beta-
lactamases)

• No activity against Gram-negative bacteria

• Exceptions:
– Spirochetes (Treponema pallidum, Borrelia
burgdorferi)
– Meningococci (some strains N. meningitidis)
 Penicillinase-Resistant Penicillins

• Methicillin, oxacillin, cloxacillin, etc.

• Resistant to enzymes penicillinases

• For the treatment of S. aureus infections,

caused by β-lactamase–producing strains

• Narrow spectrum of activity –

staphylococci
 MRSA

• Some strains S. aureus are resistant to

methicillin and oxacillin

• Called MRSA strains

• They have a mecA gene, encoding an altered

PBP

• Today MRSA take place in hospital, and

community-aquired infections
 Broad-Spectrum Penicillins
• Aminopenicillins: ampicillin, amoxicillin etc.
• Carboxypenicillins: carbenicillin, ticarcillin etc.
• Ureidopenicillins: piperacillin, mezlocillin,
azlocillin

• All 3 groups have activity mainly against Gram-

positive cocci and some Gram-negative rods

• All 3 groups are susceptible to beta lactamases

• Carboxy- and especially ureidopenicillins

have very strong activity against
Psedomonas aeroginosa
 β-Lactamase Inhibitors

• Clavulanic acid, sulbactam, tazobactam

• Relatively inactive by themselves, but can

inactivate bacterial β-lactamases

• Used in combination with some penicillins

(ampicillin, amoxicillin, ticarcillin, piperacillin)

• Effective in treating infections caused by β-

lactamase-producing bacteria
 Some Combinations
(Generic and Brand Names)

• amoxicillin and clavulanic acid –

Augmentin, Amoxiclav, etc.
• ampicillin and sulbactam – Unasyn
• piperacillin and tazobactam – Zosyn
β-Lactamase Inhibitors
 Side Effects of Penicillins
• In general have very limited toxicity
• The most severe side effects are allergic
reactions, which depend on individual
predisposition
• Penicillin is the number 1 cause of life-
threatening anaphylactic shock (a rapid and
severe allergic reaction)
• Antibiotic associated diarrhea is possible
(concerns especially broad-spectrum
penicillins)
 Cephalosporines

• Derivatives from 7-aminocephalosporanic acid

• Have the same mechanism of action as the
penicillins

• Have a wider
antibacterial spectrum

• They are resistant to

many β-lactamases
 Generations of Cephalosporins
(Cephalosporins Classes)

• The cephalosporins are classified into

several generations

• The generations reflect their

chronological development

• Each new generation covers a wide range

of antibacterial spectrum
 First-Generation Cephalosporins
(Narrow-Spectrum)

• Cefazoline, cephalexin, cephalothin, etc.

• Very active primarily against Gram-positive cocci
(except enterococci)
• Limited activity against Gram-negative bacteria
• Do not penetrate the central nervous system
• For treatment of urinary or respiratory tract
infections
 Second-Generation Cephalosporins

• Cefoxitin, cefotetan, cefamandole, cefuroxime

• Active against the organisms covered by first-

generation but have extended coverage against
Garm-negative rods (Klebsiella and Proteus,
without Pseudomonas)

• Cefoxitin and cefotetan are active against

anaerobes (Bacteroides fragilis)

• Can be used in mixed anaerobic infections

 Third-Generation Cephalosporins

• Cefoperazon, Cefotaxime, Ceftazidime, Ceftriaxone

• A major advantage is their enhanced activity against

Gram-negative rods

• Have decreased activity against Gram-positive

cocci, exept for S. pneumoniae

• Enterococci (Gram-positive) are intrinsically

resistant to third generation drugs and often
produce superinfections during their use
 Therapeutic Application of the
Third-Generation Cephalosporins
• Very useful in the management of hospital
acquired Gram-negative urinary or respiratory
tract infections or for sepsis

• Have possibility to reach the central nervous

system and to appear in the spinal fluid in
sufficient concentrations
• Examples: ceftriaxone, cefotaxime, ceftazidime

• Some antibiotics of the group - ceftazidime,

cefoperazon are active against P. aeruginosa
 Forth-Generation Cephalosporins
• Cefepime
• Have extended activity (better compared to
third generation) against Gram-positive and
Gram-negative bacteria
• Active against S. pneumoniae and methicillin-
susceptible S. aureus (MSSA)
• Additional spectrum of activity includes
genera Enterobacter, Citrobacter, P. aeruginosa
• Reserve in life-threatening infections caused
by multidrug resistant strains of Gram-
negative bacteria
 Fifth-Generation Cephalosporins

• Ceftobiprole, ceftaroline

• Широк спектър (Грам-положителни и Грам-

отрицателни бактерии)

• Active against methicillin resistant S. aureus

(MRSA)

• Additional spectrum for Listeria

monocytogenes and Enterococcus faecalis
 Side Effects of Cephalosporines

• Allergy – anaphylaxis, fever, skin rash

• Penicillins and cephalosporins have the

highest rate of allergic reaction
• Cross-allergy with penicillins - approximately 5 %

• Thrombophlebitis after intravenous injection

• Superinfections with fungi or enterococci is

possible
 Conclussion

• The third and forth-generation

cephalosporins are active against most
Enterobacteriaceae and Pseudomonas

• Have the increased susceptibility to

beta-lactamases

• Gram-negative bacteria rapidly develope

resistance to most cephalosporins, which
may significantly compromised the use of
all these agents
 Classification of Cephalosporins

First Second Third Forth Fifth

generation generation generation generation generation

Cefaclor Cefdinir
Cefamandole Cefditoren
Cefadroxil
Cefonicid Cefixime
Cefazoline
Ceforanide Cefoperazon
Cephalexin Cefepime Ceftaroline
Cefuroxime Cefotaxime
Cephaloridine
Cefprozil Cefpodoxime Cefpirome Ceftobiprole
Cephalothin Loracarbef Ceftazidime
Cephapirin Cefmetazole Ceftibuten
Cephradin Cefoxitin Ceftizoxime
Cefotetan Ceftriaxone
 Monobactams

• Aztreonam

• Narrow spectrum - only against Gram-

negative rods

• High level resistance to beta-lactamases

• Alternative to the aminoglycosides or

third generation of cephalosporins
Aztreonam
 Carbapenems

• Imipenem
• Meropenem
• Ertapenem
• Doripenem
 General Characteristics of Carbapenems

• A wide range of activity - both aerobic and

anaerobic, Gram-positive and Gram-
negative bacteria
• High level of resistance to most beta-
lactamases
• They are defined as heavy artillery against
many bacterial infections
• Extensive use for the treatment of mixed severe
infections
 Carbapenems

• Imipenem-Cilastatin (Tienam)

• Cilastatin is a peptidase inhibitor that

blocks the renal degradation of imipenem

• Meropenem – more stable to renal

dehydropeptidase I
 Carbapenemases

• During the last decade the emergence of

carbapenemase-producing strains among
Enterobacteriaceae, Pseudomonas spp.,
and Acinetobacter is remarkable
• A variety of carbapenemases have been
reported
• The resistance due to carbapenemases
compromise the efficacy of almost all beta-
lactams (except aztreonam), including the last
resort carbapenems
 Carbapenemases
• The increasing resistance to carbapenems among
Еnterobacteriaceae has become a public health
problem of major concern
• In India – the first isolated carbapenemase - the New
Delhi metallo-beta-lactamase
• In Italy, a rapid and remarkable increase of
carbapenem-resistant Klebsiella pneumoniae has
been reported since 2010
• In Central Greece, the rate of isolation of carbapenem-
resistant P. aeruginosa was around 33% during the
period 2007–2010, but a sudden increase to 50% was
observed in 2011
• In Turkey - The presence of OXA type carbapenemases
in Pseudomonas strains: first report from Turkey – 2015
 Group of Glycopeptides

• Vancomycin, teicoplanin
• Active against Gram-positive bacteria
• Inactive against Gram-negatives, molecule is
too big
• Inhibit synthesis of peptidoglycan at a site
earlier than that inhibited by beta-lactam
antibiotics
• Used for the treatment of infections, caused
by resistant strains as MRSA, Enterococcus,
as well as Clostridium difficile
 Group of Glycopeptides

• To reduce the increase in vancomycin-

resistant strains, use of this agent
should be restricted to the treatment of
very serious infections, caused by
beta-lactam resistant Gram-positive
bacteria
 Cell Wall-Active Antibiotics Used for the
Treatment of Tuberculosis

• Izoniazid, etambutol, pyrazinamid

• Clofazimine – new drug

• Different mechanisms of action – inhibition

of the mycolic acid, arabinogalactan and
specific enzymes synthesis

• The drugs are used usually in combination to

prevent development of the resistance;
prolonged treatment
INHIBITORS OF CYTOPLASMIC
MEMBRANE FUNCTION AND
STRUCTURE
 Antibiotics for Inhibition of
Cell Membrane
Function and Structure

• Lipopeptides

• Polypeptides
 Lipopeptides

• Daptomycin*
• New class - cyclic lipopeptides; approved in
2003
• Lead to membrane depolarization and
disruption

• Active against Gram-positive bacteria only

• Used for multidrug resistant bacteria:

staphylococci (MRSA), eneterococci (VRE),
streptococci (MDR S. pneumoniae)
 Polypeptides

• Polymixins – polymixin B and polymixin E (colistin)

• Active against Gram-negative bacteria (narrow
spectrum)
• Insert into the outer membrane, lead to increasing
permeability and cell death
• Historically limited use because of high level of
nephrotoxicity
• Today used for the treatment of systemic infections,
caused by Acinetobacter, Klebsiella and
Pseudomonas, susceptible only to colistin
• The last-line therapeutic options for multidrug-
resistant Gram-negative bacterial infections
 Polypeptides

• Bacitracin
• For topical usage
• Active against Gram-positive bacteria
• Gram-negative bacteria are resistant
• Inhibits the bacterial cell synthesis, may
also damage the cytoplasmic membrane
 Neutrophil Levels
• Normal level: 1500 to 7000 neutrophils per mm3 of
blood.
• Mild Neutropenia: when the ANC falls below a lower
limit of 1500 per mm3 (1.5 x109/l), but remains higher
than 1000 per mm3 (1.0x109/l).
• Moderate Neutropenia: when the ANC falls between 500
per mm3 and 1000 per mm3 (0.5x109/l – 1.0x109/l).
• Severe Neutropenia: when the ANC falls below 500 per
mm3 (0.5x109/l).
• The lower the neutrophil count, the greater the risk of
infection. This risk increases if low neutrophil counts
persist for more than three days.