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Chapter 1: Microbiology of antibiotic resistance
Most common mechanisms of Resistance of Bacteria
Antimicrobial resistance is considered to be an ancient phaenomenon (Munita & Arias,
2016) as a result of the interaction of many organisms with their environment.
Selective pressures imposed by humans have resulted in the emergence of “superbugs”,
or bacteria that are resistant to virtually all commercial antibiotics. Experts fear that
society could return to a pre-antibiotic era, when simple infections could wipe out entire
populations and surgical interventions would be life threatening (Pirnay JP, The
magistral Phage, 2018).
Since the discovery of Penicillin, antibiotic resistance has been an increasing trend
because antimicrobial compounds have also a natural origin and bacteria have evolved
to be intrinsically resistant to them; but in clinical settings, the most important
resistance is that one developed by acquisition in a colony that was prior sensitive to the
antibiotic treatment.
When a microbial species has to face a threat, it might be pushed to the selection for
random mutations in the genome that may allow survival (Goodman&Gilman, 2018).
Genetic basis of Antimicrobial resistance
Bacteria can use several strategies to adapt to antibiotics: a) mutations in genes, often
associated with the mechanism of action of the xenobiotic; b) acquisition of foreign
DNA that can code for the resistance determinants through horizontal gene transfer
(HGT) (Munita & Arias, 2016); c) hypermutable phenotypes (Goodman&Gilman,
2018).
Mutational resistance
A subgroup of bacterial cells derived from a susceptible population develops
mutations in genes that affects the activity of the drug allowing the survival of the cell.
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Once the resistant mutant emerges, the antibiotic eliminates the susceptible population
allowing the predominance of the resistant bacteria.
The genetic mutation that causes antibiotic resistance, may involve several targets all
important for the action of the antibiotic:
- Modification of the target that causes a decreasing of the affinity for
the drug;
- Decrease drug uptake through the modification of a protein involved
in the drug transport;
- Mutation of a protein important for drug activation or inactivation ;
- Changes in metabolic pathways.
HGT – horizontal gene transfer
Acquisition of foreign material is one of the most important driving forces of the
bacterial evolution and it’s often responsible for the development of antimicrobial
resistance.
Most antimicrobial agents that are used in clinical practice are derived from
natural products that are found in the environment and bacteria which share the same
environment, will develop the genetic determinants of resistance with a sort of
“environmental resistome” which is then passed through other clinically resistant
bacteria.
Bacteria can acquire foreign genetic material through a) transformation (incorporation
of naked DNA into the chromosome); b) transduction through a phage; c) conjugation
through the formation of a bacterial pilum (bacterial sex). Figure a
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Figure a (Microbiology: health and disease, 2021) describes the mechanisms through
which bacteria can acquire external genetic material.
Transformation is the simplest kind of HGT in order to acquire resistance and it consists
in the uptake and incorporation into the host genome by homologous recombination of
free DNA released in the environment by other bacterial cells. Transformation is the
molecular basis of penicillin resistance in Pneumococci and Neisseria
(Goodman&Gilman, 2018).
Conjugation is the main mechanism of resistance in the hospital which is very efficient
because it can transfer multiple resistance genes in a single move, and it involves cell-
to-cell contact through a sex pilus or bridge. Conjugation between non-pathogenic and
pathogenic bacteria it’s likely to occur at high rates in the gastrointestinal tract of
humans who are treated with antibiotics. This mechanism is common among gram-
negative bacilli; Enterococci contain a broad range of host-range conjugative plasmids
that are involved in the transfer and spread of resistance genes among gram-positive
bacteria (Goodman&Gilman, 2018).
As a general rule, conjugation uses mobile genetic elements (MGEs) as vehicles such as
plasmids and transposons.
Transduction is the acquisition of bacterial DNA from a phage that has DNA from a
previous host bacterium and it’s a common mechanism for resistance acquisition in
among strains of Staphylococcus aureus (Goodman&Gilman, 2018).
Finally, there are also integrons which represent one of the most efficient mechanism
for accumulating antimicrobial resistance genes because they are site specific
recombination system, and they are capable of recruiting open reading frames in the
form of mobile gene cassettes through their encoded integrase.
Hypermutable phenotypes
Genetic progression is guaranteed by the replicative and repair activities of DNA
polymerases and also by post replicative repair systems. When there is a defect in one
of these mechanisms, it will lead to a high degree of mutations in many genes; these
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isolates are called mutator (Mut) phenotypes and could include also mutations in genes
that cause antibiotic resistance. This mechanism of hypermutation in the DNA repair
genes, has been showed in the multidrug-resistant strains of Mycobacterium
tuberculosis Beijing phenotype (Goodman&Gilman, 2018).
Principal mechanisms of antimicrobial resistance
Over millions of years, bacteria have evolved mechanisms of drug resistance according
to which xenobiotic is used.
Example: Fluoroquinolones exert 3 different mechanisms of resistance which may
coexist in the same bacteria at a given time producing an additive effect and often
increasing the levels of resistance:
- Mutations in the genes that encode for the target site of
fluoroquinolones such as DNA gyrase and topoisomerase IV;
- Overexpression of efflux pumps that take the drug out from the cell;
- Protection of the target site by a Quinolone resistance protein (QNR).
It seems that bacteria have evolved a sort of preference for different mechanisms.
For example, the preferred mechanism of resistance to β-lactams in gram negative
bacteria, is the production of β-lactamases, while gram positive bacteria prefer to
modify the penicillin-binding protein PBP. It is thought that this may be due to the
presence of an outer membrane in gram negatives that controls the entrance of the drug
in the periplasmic space; indeed, most β-lactams require specific porins to enter and
reach the PBP as their target which is located in the inner membrane.
Gram negative bacteria hence produce the right number of β-lactamases sufficient to
keep the kinetic of the reaction toward the destruction of the antibiotic (Munita & Arias,
2016).
Modification of the antibiotic molecule
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Through the addition of chemical moieties that can inactivate the drug or make it unable
to interact with the target. This mechanism is known to be common both for gram
positive and gram negative bacteria and it’s usually reserved for antibiotics that act by
inhibiting the protein synthesis at the ribosome level.
Many enzymes are able to modify the drug through:
- Acetylation (aminoglycosides, chloramphenicol, streptogramins);
- Phosphorylation (aminoglycosides, chloramphenicol);
- Adenylation (aminoglycosides, lincosamides).
The final result is the addition of steric hindrance that decreases the capacity of
the drug to perfectly fit with the target and hence it increases its Minimum inhibitory
concentration (MIC)
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.
There is a subset of enzymes very common: the aminoglycosides modifying
enzymes- AMEs that modify covalently the hydroxyl or amino groups of the
aminoglycosides. They represent the most common mechanism of aminoglycosides
resistance worldwide; they are usually found in the chromosome of certain bacterial
species such as Enterococcus faecium and Serratia marcescens.
APH (3) family – aminoglycoside 3’ phosphotransferase- is very common for
both gram positive and gram-negative bacteria and it modifies kanamycin and
streptomycin but not gentamicin and tobramycin.
AAC (6’)-I -acetyltransferase- is mainly found in gram negative clinical isolates
that include Enterobacteriaceae, Pseudomonas spp and Acinetobacter; it targets most
aminoglycosides including amikacin and gentamicin.
The AMEs have also evolved in more than one single biochemical activity and
this is the case of AAC (6’) APH (2’’) which is found mainly in gram positive bacteria
and it’s a bifunctional enzyme (acetyltransferase+phosphotransferase) which confers
resistance toward all the aminoglycosides except for streptomycin and it’s located on a
transposon that is very common for Enterococci spp and Staphylococci spp. This
bifunctional enzyme causes the high-level resistance to gentamicin in Enterococci spp
(included the vancomycin-resistant strains) and the Methicillin-resistant S.aureus.
CATs enzymes (Chloramphenicol acetyltransferase) target chloramphenicol that
inhibits protein synthesis through the interaction with the 50S ribosomal subunit.
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The minimum inhibitory concentration is the lowest concentration capable of inhibiting the visible
growth of a microorganism after overnight incubation (Murray, Rosenthal, & Pfaller, 2009)
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Multiple copies of cat genes are present both in gram positive and in gram negative
bacteria and they are classified as type A, with high level of resistance, and type B, with
low chloramphenicol resistance; they are found both in the chromosome and plasmids
and transposons.
Destruction of the antibiotic molecule
It is a typical mechanism of resistance proper of the β-lactamases which destroy the
amide bond of the β-lactam ring, open the ring and make the antibiotic ineffective.
Infections by Penicillin-resistant Staphylococcus aureus became clinically relevant
soon after penicillin discovery and it was found out that the penicillinase was encoded
by a plasmid transmitted readily between S.aureus strains. In order to overcome this
resistance, new compounds more resistant to penicillinase were synthesised such as
ampicillin, they were designed to have also a wider spectrum. But then, a new plasmid
encoding a β-lactamase able to hydrolyse ampicillin was found among the gram-
negative bacteria, named TEM-1. From then on, new generations of β-lactams were
developed but then, new β-lactamases arose rapidly with a sort of antibiotic-driven
bacterial evolution.
The genes that encode for β-lactamases are the bla genes that can be found on
integrons, plasmids and on the chromosome, too; they can be constitutively expressed
but also inducible by the antibiotic itself.
There are more than a thousand lactamases, and they can be divided according to
Ambler classification (four classes A to D) and also through the Bush-Jacoby
classification (four categories, with several subgroups according to their substrate
specificity). These classifications do not fully overlap, for instance: Ambler class A and
D are considered in group 2 of the Bush-Jacoby.
Penicillinases able to hydrolyse penicillins, third generation cephalosporins and
monobactams but not cephamycins and carbapenems, are called ESBL (extended
spectrum β-lactamase) and most of them belongs to Ambler class A and they are mostly
inhibited by clavulanic acid or tazobactam and this distinguishes them from AmpC
enzymes that are class C lactamases and also hydrolyse third gen. cephalosporins, but
they are not inhibited by clavulanic acid or tazobactam.
A subgroup of class D OXA enzymes capable of destroying third generation
cephalosporins are also considered as ESBL.
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Another clinically relevant group of enzymes is constituted by the Carbapenemases
which are β-lactamases capable of destroying carbapenems which constitute the most
potent β-lactam available for the actual clinical practice.
Carbapenemases can be divided into serine carbapenemases (Ambler class A
and D) and metallo carbapenemases (Ambler class B) that are the most resistant ones.
Ambler class A: have a serine residue in the catalytic site (as well as class C and
D); most of them are inhibited by clavulanic acid and their spectrum of action includes
monobactams but not cephamycins (cefoxitin and cefotetan). Class A includes:
- Penicillinases: TEM-1 and SHV-1;
- ESBL: CTX-M;
- Carbapenemases: KPC (currently prevalent in several gram-negative
species).
CTX-M is a plasmid encoded ESBL which is commonly found in
K.pneumoniae, E.coli and other Enterobacteriaceae. It’s though it was acquired
through HGT from Kluyvera species
Five species of class A carbapenemases are described: 3 of them are
chromosomally encoded such as IMI (imipenem-hydrolysing enzyme), SME
(S.marcescens enzyme) and NMC (not-metallo-enzyme carbapenemases); the
other 2 are KPC and GES which are harboured in plasmids or other MGE. They
are all inhibited by clavulanic acid and tazobactam, they hydrolyze aztreonam
but not cephamycins.
Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria are a group of
emerging highly drug-resistant gram-negative bacilli causing infections
associated with significant morbidity and mortality. KPCs are an important
mechanism of resistance for an increasingly wide range of gram-negative
bacteria, no longer limited to K. pneumoniae.
KPC now is prevalently found in Klebsiella spp and in other several gram-
negative bacteria such as Enterobacteriaceae species, E. coli, Proteus mirabilis,
Salmonella spp and Pseudomonas Aeruginosa.
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Ambler class B: they are metallo β-lactamases because they use a metal ion
(usually zinc) as a cofactor -instead of a serine residue- for the nucleophilic attack on
the β-lactam ring. They are inhibited by chelating agents such as EDTA and are active
toward β-lactams, including carbapenems. They are not inhibited by clavulanic acid or
tazobactam, they hydrolyze cephamycins but not aztreonam.
Class B include:
- IMP, firstly described in Japan from S.marcescens and since then also
in Enterobacteriaceae, Pseudomonas, Acinetobacter. BlaIMP genes are
found on large plasmids and forming part of class 1 integrons.
- VIM firstly described in Verona (Verona integrons-encoded metallo
β-lactamase), they were found in P.aeruginosa spp and now it’s
widely distributed among different species of bacteria. VIM-2 is the
one widely distributed all over the world.
- NDM-1 firstly described in a K.pneumoniae isolate from a New
Delhi patient cured in Sweden (New Delhi metallo β-lactamase). Its
genes are plasmid encoded and they are readily transferrable to other
gram-negative organisms.
It has been found that the MGEs that contains the genes for NDM
carry other multiple resistance genes such as carbapenemases, ESBL,
AMEs and methylases. This confers the phenotype of MDR
pathogen. And the worldwide diffusion of the NDM-1 gene
represents an issue for the clinical settings of infections and bacteria
producing NDM-1 has also been found in the soil and drinking water
stating that these genes might be integrated in the human microbiota.
This, actually, represents the real threat because in order to treat this
kind of infection, we must use more dangerous and toxic antibiotics
which are not commonly used in the clinical practice.
Ambler class C: they confer resistance to all penicillins and to cephalosporins,
including cephamycins. They don’t hydrolyze aztreonam and they are not inhibited by
clavulanic acid. The most relevant is AmpC, generally encoded on the chromosome by
Enterobacter spp., Citrobacter freundii, S.marcescens, Morganella spp, P.aeruginosa
but Proteus spp. and Klebsiella spp do not harbour AmpC in their chromosome.
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