Revisiting Antibiotic Resistance Mechanistic

antibiotics
Review
Revisiting Antibiotic Resistance: Mechanistic Foundations to
Evolutionary Outlook
Chowdhury M. Hasan 1,2,3, * , Debprasad Dutta 2,4 and An N. T. Nguyen 3
1 School of Biological Sciences, University of Queensland, Brisbane 4072, Australia

2 Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary &
Ecological Sciences (IVES), University of Liverpool, Liverpool L7 3EA, UK; [email protected]
3 School of Biological Sciences, Monash University, Melbourne 3800, Australia; [email protected]
4 Department of Human Genetics, National Institute of Mental Health & Neurosciences (NIMHANS),
Bangalore 560029, India
* Correspondence: [email protected]
Abstract: Antibiotics are the pivotal pillar of contemporary healthcare and have contributed towards
its advancement over the decades. Antibiotic resistance emerged as a critical warning to public
wellbeing because of unsuccessful management efforts. Resistance is a natural adaptive tool that
offers selection pressure to bacteria, and hence cannot be stopped entirely but rather be slowed down.
Antibiotic resistance mutations mostly diminish bacterial reproductive fitness in an environment
without antibiotics; however, a fraction of resistant populations ‘accidentally’ emerge as the fittest
and thrive in a specific environmental condition, thus favouring the origin of a successful resistant
clone. Therefore, despite the time-to-time amendment of treatment regimens, antibiotic resistance has
evolved relentlessly. According to the World Health Organization (WHO), we are rapidly approach-
ing a ‘post-antibiotic’ era. The knowledge gap about antibiotic resistance and room for progress is
evident and unified combating strategies to mitigate the inadvertent trends of resistance seem to
be lacking. Hence, a comprehensive understanding of the genetic and evolutionary foundations of

antibiotic resistance will be efficacious to implement policies to force-stop the emergence of resistant
Citation: Hasan, C.M.; Dutta, D.; bacteria and treat already emerged ones. Prediction of possible evolutionary lineages of resistant bac-
Nguyen, A.N.T. Revisiting Antibiotic teria could offer an unswerving impact in precision medicine. In this review, we will discuss the key
Resistance: Mechanistic Foundations molecular mechanisms of resistance development in clinical settings and their spontaneous evolution.
to Evolutionary Outlook. Antibiotics
2021, 11, 40. https://doi.org/
Keywords: antibiotic resistance; bacteriostatic; bactericide; evolution; adaptation; epistasis; drug
10.3390/antibiotics11010040
interaction; mutant selection window; compensatory evolution; clonal interference
Academic Editor: Jordi Vila
Received: 31 August 2021

Accepted: 23 November 2021
Published: 30 December 2021
1. Introduction
The introduction of antimicrobials to treat infectious diseases was one of the greatest
Publisher’s Note: MDPI stays neutral medical achievements in history [1]. The objective was to save millions of lives facing
with regard to jurisdictional claims in severe infectious diseases caused by various pathogens, including bacteria, fungus, viruses,
published maps and institutional affil-
and parasites. Though these antimicrobial drugs have saved millions of lives, the global
iations.
emergence of resistance mechanisms among these pathogens has seriously undermined
our current treatment options [2,3]. Therefore, treatment failure due to the emergence of
antimicrobial resistance is now a global public health threat and a tremendous economic
risk [4,5]. Among these pathogens, bacteria are the most striking example in terms of
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
morbidity and mortality. The worrying picture is that by 2050 global death due to an
This article is an open access article
increase in antibiotic resistant bacteria is estimated to be 50 million per year at an increasing
distributed under the terms and
global cost of $100 trillion USD [6]. Bacteria exploit various efficient strategies to neutralize
conditions of the Creative Commons the action of antibiotics, often leaving no effective treatment options that can be employed
Attribution (CC BY) license (https:// to treat infectious diseases caused by them. It is well evident that bacteria employ various
creativecommons.org/licenses/by/ physiological and biochemical mechanisms to develop tolerance or resistance [7]. This
4.0/).
Antibiotics 2021, 11, 40. https://doi.org/10.3390/antibiotics11010040 https://www.mdpi.com/journal/antibiotics

Antibiotics 2021, 10, x FOR PEER REVIEW 2 of 23
Antibiotics 2021, 11, 40 2 of 23
action of antibiotics, often leaving no effective treatment options that can be employed to
treat infectious diseases caused by them. It is well evident that bacteria employ various
review will highlight types of antibiotics, their mechanisms of action, and the mechanistic
physiological and biochemical mechanisms to develop tolerance or resistance [7]. This re‐
and evolutionary
view basis
will highlight underlying
types resistance
of antibiotics, evolution in
their mechanisms of bacterial populations.
action, and the mechanistic
and evolutionary basis underlying resistance evolution in bacterial populations.
2. Antibiotics and Their Impact on Bacterial Cellular Perturbation
The discovery
2. Antibiotics of antibiotics
and Their Impact on has drastically
Bacterial changed
Cellular modern medicine and extended
Perturbation
the human lifespan. The first naturally occurring antibiotic,
The discovery of antibiotics has drastically changed modern namely penicillin,
medicine was discov-
and extended
ered Fleming from Penicillium notatum
the human lifespan. The first naturally occurring antibiotic, namely penicillin, was dis‐ into
by Alexander in 1928, which was introduced
clinical
covered practice in 1941Fleming
by Alexander [8,9]. Most
fromofPenicillium
the antibiotics
notatumthat
in are used
1928, whichtoday
waswere discovered
introduced
and
intointroduced into the
clinical practice market
in 1941 [8,9].before
Most of1987, and this period
the antibiotics that areisused
often termed
today wereasdiscov‐
the golden
decade
ered and [10]. Particularly,
introduced themarket
into the cessation of new
before 1987, antimicrobial
and this periodclasses
is oftenwas apparent
termed as the from
golden
1987 untildecade
today[10]. Particularly,
[11]; during thisthe cessation
period, onlyof a
new
fewantimicrobial classes was apparent
(~9) new antimicrobial classes were
from 1987 until
discovered today [11];into
and launched during
thethis period,(Figure
markets only a few (~9) new
1). With thisantimicrobial
slow rate of classes
new antibi-
were
otic discovered
discovery andand
thelaunched into the markets
rapid emergence (Figure
of resistant 1). With
bacteria, wethis
areslow rate
in the of new
post-antibiotic
antibiotic
era [12]. discovery and the rapid emergence of resistant bacteria, we are in the post‐an‐
tibiotic era [12].
Figure1.1. Historical
Figure Historical panorama
panorama ofof
antibiotic launch
antibiotic and resistance
launch detection.
and resistance The x‐axis
detection. Theindicates
x-axis indicates
different types of antibiotics and the corresponding y‐axis shows the year of introduction into clin‐
different types of antibiotics and the corresponding y-axis shows the year of introduction into clinical
ical practices. Resistance histories to different antibiotics are shown by different circles. Connect‐
practices. Resistance
ing line between emptyhistories to different
and filled coloured antibiotics
circles showsarethe
shown
year ofbyintroduction
different circles. Connecting
of a specific anti‐ line
between empty
biotic into and
clinical filled coloured
practice circles
and the year shows the
of resistance year offor
observed introduction of aeach
that antibiotic; specific antibiotic into
coloured
circle further
clinical practicerepresents
and the different bacterial species.
year of resistance observedFor example, colistin waseach
for that antibiotic; firstcoloured
introduced into further
circle
clinical practice
represents in 1952
different [13], but
bacterial resistance
species. to colistin colistin
For example, was firstwas
reported in clinical P.into
first introduced aeruginosa and
clinical practice
in 1952 [13], but resistance to colistin was first reported in clinical P. aeruginosa and K. pneumoniae
(shown by a specific coloured circle) in 1998 [14]. Penicillin resistant laboratory E. coli was reported
in 1950 [15] before its introduction into clinical practice in 1941, but the first penicillin resistance
clinical S. aureus was reported in 1942 [16,17]. PDR: pan-drug resistant; VR: vancomycin resistant;
spp: species; ND: resistance mechanism not detected.
Antibiotics belong to different classes of chemicals—including those of biological,

synthetic, or semi-synthetic origin—and have selective modes of action. Based on their
mechanisms of action, antimicrobial compounds are classified into two groups: bacte-
riostatic (Table 1) and bactericidal (Table 2). Bacteriostatic drugs are only able to inhibit
or hinder growth but cannot kill bacteria. Whereas drugs are called bactericidal when
exposure to this group of antibacterial compounds leads to the death of bacteria. Both
drugs principally interfere with bacterial cell-wall biosynthesis, DNA synthesis, RNA
synthesis, and protein synthesis [18]. Furthermore, antibiotics have been classified based
on the cellular components or systems they affect in bacteria. Some of these antimicro-
bial agents target the synthesis of important cellular components, whereas some other
classes of antibiotics interfere with bacterial nucleic acid synthesis or repair [19,20]. The
mechanistic actions of the bacteriostatic and bactericidal are featured in Tables 1 and 2.
Furthermore, we have described the mechanistic action of the major bactericidal antibiotics
in the following sections.
A commonly used bactericidal class of antibiotic is fluoroquinolone which targets and
inhibits DNA replication by interfering with topoisomerase II enzyme—also known as
DNA gyrase composed of two subunits encoded by gyrA and gyrB—leading to cellular
death by forming double-strand DNA breaks [21]. Whereas beta-lactam antibiotics—
including penicillins, cephalosporins, carbapenems, and monobactams—act by binding
to and inhibiting the penicillin binding proteins (PBP) leading to stop in cross-linking
or transpeptidations within the bacterial cell wall, and thus undergo cellular death [22].
Furthermore, bactericidal antibiotics interfere with common metabolic systems such as
the central metabolic pathways called tri-carboxylic acid (TCA) cycle and iron metabolism.
For example, reactive oxygen radicals in response to lethal bactericidal antibiotics result in
cellular death [23,24].
Table 1. Action and common resistance mechanisms of major bacteriostatic antibiotics.
Bacteriostatic Candidates Mode of Action Mechanism of Resistance

Reversibly inhibits 30S ribosomal subunit
Tetracycline Efflux system and protecting ribosomes [26].
of bacteria [25].
Macrolides Methylation of the 23S rRNA, efflux system [28].
of bacteria [27].
Inhibits folate synthesizing enzyme By horizontal transfer of dihydropteroate synthase
Sulphonamides
dihydropteroate synthase (DHPS) [29]. gene [30].
Acetyltransferases vatD gene expression mediates
Streptogramins streptogramin A, wheras vatE and ermB or vgbA gene
of bacteria [31].
cluster confers streptogramin B antibiotics [32].
Oxazolidinones High diversity and coselection of optrA [34].
of bacteria [33].
Reversibly inhibits 50S ribosomal subunit Target site modification, efflux system and drug
Lincosamides
of bacteria [35]. inactivation [36].
Occupying the active site of bacterial
Increase expression of DHFR or decrease the affinity of
Trimethoprim dihydrofolate reductase (DHFR), thus
DHFR to the drug [38].
blocking the activity of the enzyme [37].
Table 2. Action and common resistance mechanisms of major bactericidal antibiotics.
Bactericide
Mode of Action Mechanism of Resistance
Candidates
Competitively inhibits the transpeptidase Beta-lactamase encoded by blaZ, altered PBP2a encoded by
Penicillins enzyme resulting cross-linking blockage in mecA [14,40], extended-spectrum-beta-lactamases (ESBLs),
cell wall [39]. AmpC beta-lactamase (i.e., blaAmpC) [41–43].
Competitively inhibits the transpeptidase
AmpC beta-lactamase (i.e., blaAmpC), ESBLs (i.e.,
Cephalosporins enzyme resulting in cross-linking or blockage
blaCTX-M) [41,42].
in cell wall [44].
Carbapenemases (i.e., class A serine-carbapenemase
including KPCs; class B metallo-carbapenemase including
New-Delhi-metallo-beta-lactamases or NDM,
Verona-integron-encoded beta-lactamases or VIM,
Binding with penicillin-binding proteins
Imepenemase IMP-carbapenemase (also a
Carbapenems (PBPs) and inactivation of these proteins
metallo-beta-lactamase); class D serine carbapenemase such
leads to cell wall synthesis interruption [45].
oxacillinase (OXA) [46,47], mutation-derived target enzyme
modification [48]; preventing the drug entry by modifying
outer membrane permeability [49]; pumping carbapenems
out by efflux pump systems [50].
Mostly through aminoglycosides modifying enzymes
Binding with 30 s ribosomal subunit
encoded by aac (aminoacetyl-tranferase) and aph
Aminoglycoside resulting genetic code misreading followed
(aminophospho-transferase), efflux system, or mutation in
by interruption of bacterial translation [51].
rpsL and 16S rRNA [43,52].
Target enzyme mutation (DNA gyrase encoded by gyrA and
Interrupting bacterial DNA replication by
Fluoroquinolones gyrB, and topoisomerase IV encoded by parC and parE
inhibiting topoisomerases [53].
genes), efflux system and changing drug entry [54].
Interrupting transcription by inhibiting Mutation of the target (beta subunit of RNA polymerase
Rifamycin
bacterial RNA polymerase [55]. encoded by rpoB) [56].
The pmrHFIJKLM (also known as arn operon) and pmrCAB
operon—both invove in the biosynthesis of LAra4N and
modify the lipid A, thus disrupt lipid A charges [17];
Binding to lipid A of LPS and interfere with
Polymyxins mutations in genes encoding the two-component regulatory
outer membrane permeability [57].
systems such as pmrAB [58], phoPQ and plasmid-borne mcr
genes confer resistance to colistin—the last line of
drug [59,60].
Binding to anionic phospholipids in the Mutations in gene mprF which encodes the multiple peptide
Daptomycin
cytoplasmic membrane [61]. resistance factor [62].
Binding to the dipeptide terminus
D -Ala- D -Ala
of peptidoglycan pentapeptide
Replacing D-Ala-D-Ala with D-Ala-D-lac or D-Ala-D-Ser
Vancomycin precursors preventing peptidoglycan
alternatives to which vancomycin has low affinity [64].
crosslinking leads to the inhibition of
bacterial cell wall synthesis [63].
Rifamycin is a semi-synthetic bactericidal class of antibiotic that can induce cell death
by inhibiting bacterial RNA synthesis [65]. During the execution of the normal cellular
function, beta-subunit of DNA-dependent RNA polymerase enzyme is involved in the
stable channel formation between RNA-polymerase and DNA complex from which newly
synthesized RNA strand arises [66,67]. Rifampicin stably binds to the beta-subunit of
DNA dependent RNA polymerase (encoded by rpoB gene), thus inhibiting the high-fidelity
transcription and causing cellular death.
Another class of bactericidal antibiotic such as aminoglycoside causes bacterial cellular
death by interfering with cellular energetics, ribosome binding and protein synthesis
inhibition [68]. Bacterial protein synthesis through the translation of mRNA occurs in
a sequential fashion involving initiation, elongation, and termination. This process is
operated in the cytoplasmic space involving the collaborative action of the ribosome
(which acts as a factory) and many other important accessory translation factors available
in the cytoplasm [69]. The ribosome is composed of two ribonucleoprotein subunits called
the 30S (encoded by rpsL gene) and 50S. Following the formation of a complex between
mRNA-transcript, N-formyl methionine-charged aminoacyl tRNA, several initiation factors
and a free 30S subunit (this process is called initiation step of translation), the ribosome is
assembled for the next translational step [70]. While translation is a complex process that
requires many cellular components and translation factors, drugs can interfere with protein
synthesis in various ways. Antibiotics that inhibit protein synthesis are classified into the
50S and 30S inhibitors, respectively. The 50S inhibitors (i.e., erythromycin, clindamycin,
streptogramin, chloramphenicol, and linezolid) interfere with protein synthesis by blocking
the initiation of protein translation or translocation of peptidyl tRNAs [71,72]. Inhibition
of 30S ribosome (i.e., caused by tetracyclines and aminocyclitols) involves blocking of the
access of aminoacyl tRNAs to the ribosome. Both spectinomycin and aminoglycosides—
including streptomycin, kanamycin, and gentamycin—bind to the 16S rRNA, a component
of the 30S ribosomal subunit. Specifically, aminoglycosides bind to the 16S rRNA, which
in turn alter the conformation of the complex formed between an mRNA codon and its
cognate charged aminoacyl tRNA in the ribosome. This interaction results in defective
protein [19,26,73], thus cellular death occurs.
Polymyxins group of antibiotics (polymyxin A–E) are of cationic cyclic polypeptide
origin with strong bactericidal effects. Among these, only polymyxin B and polymyxin
E—also called colistin—are used in clinical practices [74]. Both antibiotics are commonly
prescribed to treat infections caused by Gram-negative bacterial pathogens, particularly
against extensively drug resistant (XDR) P. aeruginosa and A. baumannii [75]. These cyclic
antimicrobial peptides have long hydrophobic tails which directly interact with LPS of the
outer membrane of Gram-negative bacteria. Particularly, polymyxins destabilize calcium
and magnesium bridge by binding to the lipid A component of the LPS. This event causes
bacterial outer membrane permeabilization and allows polymyxins to enter the outer
membrane, leading to cellular death [60].
Daptomycin is another class of bactericidal antibiotic and is composed of cyclic
polypeptides. Daptomycin is used for the treatment of infections caused by Gram-positive
bacteria, particularly against methicillin resistance S. aureus and Enterococcus faecium. Dap-
tomycin interacts with anionic phospholipids in the presence of calcium ions in the cyto-
plasmic membrane. This interaction helps daptomycin penetrate the membrane, which
ultimately causes membrane depolarization and cellular death. However, unlike most
other bactericidal antibiotics, the detailed mechanistic basis of cellular death and resistance
for daptomycin is not yet fully elucidated [76]. Detailed mechanisms of action of the major
classes of bactericidal antibiotics are depicted in Figure 2.
Figure 2. Diversity of antibiotic resistance mechanisms. The figure shows the major bactericidal
Figure 2. Diversity of antibiotic resistance mechanisms. The figure shows the major bactericidal
antibiotics and their different targets. Beta-lactam antibiotics degrade bacterial cell wall by interfering
antibiotics and their different targets. Beta‐lactam antibiotics degrade bacterial cell wall by inter‐
with cross-linking
fering or transpeptidations
with cross‐linking within the
or transpeptidations bacterial
within cell wall cell
the bacterial by binding
wall by with PBPwith
binding (panel
PBPA),
aminoglycoside interferes with protein synthesis by binding with 30S ribosomal subunit
(panel A), aminoglycoside interferes with protein synthesis by binding with 30S ribosomal subunit (panel B),
rifamycin
(panel B),inhibits bacterial
rifamycin transcription
inhibits by interfering
bacterial transcription by with beta-subunit
interfering of DNA dependent
with beta‐subunit of DNA RNA
de‐
pendent RNA
polymerase polymerase
enzyme (panel enzyme (panel
C), whereas C), whereas
quinolone quinolone
class class of
of antibiotics antibiotics
inhibit DNA inhibit DNA
synthesis by
synthesis by
interfering interfering
with with DNA topoisomerase
DNA topoisomerase (panel D). OM: (panel
outerD). OM: outerPGL:
membrane; membrane; PGL: pepti‐
peptidoglycan layer;
doglycan
IM: layer; IM: inner
inner membrane; PBP:membrane;
penicillin PBP: penicillin
binding protein.binding protein.ofMechanism
Mechanism of action of and
action of polymyxin pol‐
ymyxin and daptomycin is
daptomycin is provided in the text.provided in the text.
3.3.Bacteria
BacteriaEmploy
EmployDiverse
DiverseDevices
DevicestotoResist
ResistAntibiotics
AntibioticsandandSpread
Spread Antibiotic Re‐
Antibiotic
sistance Resistance
Antibiotic
Antibioticresistance
resistancecancanbebedefined
definedasasthe
theproperty
propertyinherent
inherentininbacteria
bacteriabybywhich
which
successful uses of antibiotics are compromised by the development of tolerance
successful uses of antibiotics are compromised by the development of tolerance or re‐ or resistance
against them
sistance [7,77].
against Generally,
them antibiotic resistance
[7,77]. Generally, antibiotic is associated
resistance with prolonged
is associated exposure
with prolonged
toexposure
antibiotics.
to antibiotics. More specifically, a bacterial population remains susceptibleatto
More specifically, a bacterial population remains susceptible to antibiotics
the
antibiotics atof
beginning treatment
the beginningbutofcan sustain and
treatment but develop resistance
can sustain againstresistance
and develop these antibiotics
against
gradually. Therefore, the continuous selective pressure exerted by antibiotics
these antibiotics gradually. Therefore, the continuous selective pressure exerted by anti‐ has helped
bacteria develop
biotics has helped resistance
bacteriatodevelop
one or more drugstosimultaneously
resistance one or more drugs [78]. simultaneously
In recent years, the
[78].
types and mechanisms of antibiotic resistance have become more complex
In recent years, the types and mechanisms of antibiotic resistance have become due to more
the rapid
com‐
emergence
plex due to of the
newrapid
mechanisms
emergence in bacterial populations.in
of new mechanisms Innate or intrinsic
bacterial resistance
populations. andor
Innate
acquired resistance are two major types of antibacterial resistance mechanisms employed by
intrinsic resistance and acquired resistance are two major types of antibacterial resistance
bacteria to inhibit the lethal effects of antibiotics. Intrinsic resistance is a naturally occurring
mechanisms employed by bacteria to inhibit the lethal effects of antibiotics. Intrinsic re‐
trait which refers to bacterial ability to withstand the lethal effects of antibiotics due to its
sistance is a naturally occurring trait which refers to bacterial ability to withstand the le‐
structural or functional features. Therefore, intrinsic resistance is not related to previous
thal effects of antibiotics due to its structural or functional features. Therefore, intrinsic
antibiotic exposure or horizontal gene transfer. Gram-positive bacteria are susceptible

to various antibiotics because of their inbuilt architecture of peptidoglycan, which offers
large permeability of various antibiotics. In Gram-negative bacteria, the outer membrane
(lipopolysaccharide, LPS) acts as a protective filter and provides intrinsic resistance to
many antibiotics. Efflux pumps of Gram-negative bacteria also provide natural resistance
to many antibiotics [79]. In the following sections, we will discuss more about intrinsic and
acquired resistance to various antibiotics.
3.1. Natural Defense Systems to Antibiotics

Naturally occurring genes in the chromosome of the bacterial host confer intrinsic
resistance. All bacterial species exhibit intrinsic resistance to specific antimicrobial classes.
The biology of these mechanisms differs among bacteria. Some bacteria confer resistance by
increased expression of efflux pump systems, which drive antibiotics out of the cells before
they reach the target location, whereas others lack appropriate transport systems. As a
result, antibiotics cannot penetrate the cell wall and reach the target spots. Several bacteria
have genes or enzymes that confer innate resistance to antibiotics. Streptomyces, for exam-
ple, has genes that confer resistance to streptomycin, which it produces. Beta-lactamase
and inducible AmpC cephalosporinases, which hydrolyze beta-lactam antibiotics in the
periplasmic space and are carried by Gram-negative bacteria, are other examples [80].
Another type of intrinsic resistance commonly observed in bacteria is impermeability. Im-
permeability occurs when antimicrobial chemicals are unable to enter through the bacterial
outer membrane. For example, vancomycin can only target peptidoglycan crosslinking in
Gram-positive bacteria by binding to the D-ala-D-ala peptide chain; however, vancomycin
cannot penetrate through the outer membrane and reach peptides in the periplasm in
Gram-negative bacteria [81]. Bacterial intrinsic mechanism of resistance via efflux pump
system is a significant resistance mechanism, predominantly observed in Gram-negative
bacteria. While an antibiotic can pass through a membrane-spanning porin protein called
outer membrane protein (OMP) and reach the periplasmic area, it is eliminated or pumped
out of the periplasm by many active efflux mechanisms. For example, intrinsic resistance
to tetracycline, chloramphenicol and norfloxacin is well evident in Gram-negative P. aerugi-
nosa, and this process is mediated by chromosomally encoded MexAB-OprM efflux system.
Figure 3 portrays different examples of inherent resistance mechanisms. For example,
a beta-lactam antibiotic (i.e., depicted as antibiotic A) that targets a penicillin-binding
protein (PBP) channels through the outer membrane proteins (porins) and binds to the PBP
target site, interfering with bacterial cell wall synthesis—a natural mechanism of action for
beta-lactam antibiotics. Whereas antibiotic B (aminoglycoside antibiotic) can pass through
the porin channel but is efficiently pumped out of the periplasm via the efflux pump. There
are some antibiotics, such as polymyxins class (i.e., portrayed as antibiotic C) cannot cross
the bacterial outer membrane [82].
3.2. Bacteria Acquire High Levels of Antibiotic Resistance through De Novo Mutations and
Horizontal Gene Transfer
Bacteria can acquire resistance through spontaneous mutations, which result from
errors in the cellular and metabolic processes such as DNA replication, transcription,
recombination [83]. For instance, mutations in the target genes alter the binding site of
antibiotics, or mutations at the target sites can lead to the overexpression of targets during
the transcriptional step. These targets otherwise are naturally expressed at a very low
level. In some instances, specific mutations can modify the drug targets. As a result, the
minimum inhibitory concentration (MIC) of a particular antibiotic increases beyond the
therapeutic limit [84]. In Enterobacter sp. and P. aeruginosa, overexpression of the blaAmpC
is caused by mutation in the regulatory gene. In the presence of AmpC cephalosporinases,
this overexpressed gene disrupts enzyme-to substrate ratio, consequently resistance to both
penicillin and extended-spectrum cephalosporin occurs [85]. Other resistance mechanism
such as mutation in penicillin binding protein 2b (pbp2b) gene can result in penicillin
resistance in Pneumococci, whereas mutation in gene encoding ribosomal protein (i.e., rpsL)
eliminated or pumped out of the periplasm by many active efflux mechanisms. F
ple, intrinsic resistance to tetracycline, chloramphenicol and norfloxacin is well e
Gram‐negative P. aeruginosa, and this process is mediated by chromosomally
Antibiotics 2021, 11, 40 MexAB‐OprM efflux system. Figure 3 portrays different examples of8 of inherent
23 r
mechanisms. For example, a beta‐lactam antibiotic (i.e., depicted as antibiotic A)
gets a penicillin‐binding protein (PBP) channels through the outer membrane
(porins)
triggers and conformational
altered binds to the PBP target
change site,
for the interfering
drug-target with
binding bacterialwhich
interaction cell wall
in synt
turn confers resistance to different aminoglycoside antibiotics. Altered efflux
natural mechanism of action for beta‐lactam antibiotics. Whereas antibiotic B (am systems
mediated by many different mutations can also cause up-regulation or overexpression
coside antibiotic) can pass through the porin channel but is efficiently pumped o
of the efflux systems, therefore antibiotics cannot reach to the target sites of the bacterial
periplasm
cells via the efflux
[85]. Fluoroquinolone pump.E.There
resistant are
coli and some antibiotics,
Pseudomonas aeruginosasuch
are theastwo
polymyxins
best c
portrayed as antibiotic C) cannot
examples of this type of resistance. cross the bacterial outer membrane [82].
Figure
Figure 3. Active efflux
3. Active pumping
efflux systemsystem
pumping to eliminate antibiotics antibiotics
to eliminate from the periplasm.
from theEfflux pump Efflu
periplasm.
is associated with intrinsic antibiotic resistance. Intrinsic resistance is considered as phenotypic
is associated with intrinsic antibiotic resistance. Intrinsic resistance is considered as pheno
resistance as tolerance is not mediated by any genetic mutation. A = beta-lactam antibiotic which
resistance as tolerance is not mediated by any genetic mutation. A = beta‐lactam antibiotic
binds to the penicillin binding protein (PBP) and destabilizes peptidoglycan; B = aminoglycoside
antibiotic; C = polymyxin antibiotic. Most notably, decreased susceptibility mediated by efflux system
is mostly linked with aminoglycosides and fluoroquinolone, which is predominantly observed in
Gram-negative bacteria.
Bacteria can also develop resistance via the acquisition of resistance genes or resistance
determinants externally. This process is known as horizontal gene transfer (HGT). HGT can
occur through three main mechanisms: transduction, transformation and conjugation [86],
as shown in Figure 4. Furthermore, new processes such as nanotubes [87] or extracellular
vesicles mediated HGT have recently been reported [88]. During the transduction process,
resistance gene transfer is mediated by bacteriophage which infects and transfers resistance
genes to new bacterial species as witnessed in methicillin-resistant Staphylococcus aureus
(MRSA) which developed resistance through the acquisition of resistance-conferring mecA
gene from other bacterial species by transduction [89]. In another example, resistance genes
from the dead bacterial DNA can be taken up and integrated into the recipient’s chromo-
somes by homologous recombination through a process called natural transformation. Ac-
quired resistance by natural transformation is assumed to have frequently occurred in many
clinical bacterial species. For example, Streptococcus pneumoniae is thought to have acquired
penicillin-binding proteins (PBP2Bs) from the dead Streptococcus mitis [90], whereas the
acquisition of the ceftriaxone resistance penA gene in Neisseria gonorrhoeae [91] was acquired
through natural transformation. Lastly, bacteria acquire resistance genes or genetic deter-
minants by conjugation which is mainly mediated by mobile genetic elements (MGEs) such
as different types of plasmids and transposons [92,93]. The structure of MGEs is illustrated
in Figure 5. To date, most of the resistance genes that disseminate to important clinical
Gram-negative bacteria are of plasmid-borne conjugative resistance determinants ori-
gins. Among these, the notorious carbapenemase hydrolyzing enzymes in Gram-negative
Enterobacteriaceae and in others Gram-negative bacteria have become the major global
health threats because resistance genes harbouring plasmids can be rapidly disseminated
to other susceptible bacteria through conjugation [94]. Particularly, conjugation-based
plasmid-mediated antimicrobial resistance poses a serious threat to human health. Because
enzymes (i.e., KPC carbapenemase and NDM-1 metallo-beta-lactamases) that hydrolyze
plasmids [95,96]. Transmission of resistance genes between plasmids and bacterial chro‐
mosomes via integrative chromosomal elements (ICEs) is also mediated by conjugation
process (Figure 5). This mechanism of ICE‐mediated resistance propagation mostly prev‐
alent in Gram‐negative bacteria, but in some instances, this can also be found in Gram‐
positive bacteria, as reported in Streptococci spp. [97,98].
carbapenems—the last resort antimicrobial class against Gram-negative bacteria—as well
as other beta-lactams were found on different plasmids [95,96]. Transmission of resistance
plasmids
genes [95,96].
between Transmission
plasmids of resistance
and bacterial genes between
chromosomes Bacteriophage
via plasmids andchromosomal
integrative bacterial chro-elements
Transduction
mosomes via integrative chromosomal elements (2)
(ICEs) is also mediated by conjugation
(ICEs) is also mediated by conjugation process (Figure 5). This mechanism of ICE-mediated
process (Figure 5). This mechanism of ICE-mediated resistance propagation mostly prev-
resistance propagation mostly prevalent in Gram-negative bacteria, Plasmidbut harbouring
in some instances,
Transformation
alent
this
in Gram-negative (1)
bacteria, but in some instances, this can also be found in Gram-
can also be found in Gram-positive bacteria, as reported in Streptococcigene
spp. [97,98].
positive bacteria, as reported in Streptococci spp. [97,98]. resistance
Bacteriophage Conjugation (3)

Transduction (2) Donor cell
Donor cell
Transformation (1)
Plasmid harbouring
resistance gene
Resistance gene containing Conjugation (3)Donor cell
Donor cell
genetic material
Donor cell
Resistance gene containing Donor cell

genetic material
Recipient cell
Figure 4. Trinity of horizontal resistance gene transfer modalities. Transmission of genetic material
by horizontal genetic transfer, which is accomplishedRecipient cell by three different mechanisms: transfor‐
mation—bacteria take up naked DNA from the environment and integrate it to their chromosomes
Figure 4. Trinity of horizontal resistance gene transfer modalities. Transmission of genetic material
Figure
(1), 4. Trinity of horizontal resistance
transduction—bacteriophages gene
carry transfer modalities.
resistance genes andTransmission
transfer them of genetic material
to multiple by (2),
hosts
by horizontal genetic transfer, which is accomplished by three different mechanisms: transfor-
horizontal
and genetic
mation—bacteria transfer,
conjugation—resistance
take which
up naked DNA isfrom
genes accomplished
arethe by three
transferred
environment anddifferent
between mechanisms:
bacterial
integrate cells
it to their transformation—
through
chromosomes cell‐to‐cell con‐
bacteria
(1), take up naked DNA
transduction—bacteriophages
tact (3). from
carry the environment
resistance genes andand integrate
transfer them toit to their
multiple chromosomes
hosts (2), (1),
and conjugation—resistance genescarry
transduction—bacteriophages are transferred
resistancebetween bacterial
genes and cells through
transfer them tocell-to-cell
multiple con-
hosts (2), and
tact (3).
conjugation—resistance genes are transferred between bacterial cells through cell-to-cell contact (3).
Figure 5. Antibiotic resistance via acquisition of mobile genetic elements (MGEs). The structure of
a resistance plasmid (R100) (panel A) and the process of resistance gene acquisition (panel B) are
illustrated. Resistant plasmid harbouring many different resistance genes as part of transposon
Figure 5. Antibiotic
(Tn) element resistance
can confer multidrugvia acquisition
resistance of mobile
by a single geneticevent.
elements (MGEs). The structure of
Figure 5. Antibiotic resistance via acquisition of conjugation
mobile genetic Integron
elements mediated
(MGEs). The structure of
aresistance
resistance plasmid
gene capture(R100)
system(panel A) and
is frequently the process
observed of different
in many resistance genebacterial
clinical acquisition (panel B) are
species.
a resistance plasmid (R100)
Integrase (transcribed
(panel A) promoter
and the process of resistance gene acquisition (panel B) are
illustrated. Resistantunder a downstream
plasmid harbouring many(Pint)) catalyzes
different the insertion
resistance genesofasanpart
integron.
of transposon
illustrated. Resistant plasmid harbouring many different resistance genes as part of transposon
(Tn) element can confer multidrug resistance by a single conjugation event. Integron mediated
(Tn) element can confer multidrug resistance by a single conjugation event. Integron mediated
resistance gene capture system is frequently observed in many different clinical bacterial species.
resistance gene capture system is frequently observed in many different clinical bacterial species.
Integrase (transcribed under a downstream promoter (Pint)) catalyzes the insertion of an integron.
Antibiotics 2021, 11, 40 10 of 23
Integrase (transcribed under a downstream promoter (Pint)) catalyzes the insertion of an integron.
Resistance gene cassette 1 (blue) is integrated into the attI site, which is under the influence of an
upstream promoter (Pant). This way, many different resistance genes can be captured repeatedly
for example, resistance gene 2. All resistance genes remain under the same promoter and thus
become a resistance operon. Tn: transposon; bp: base-pair; tet: tetracycline resistance gene; cat:
chloramphenicol acetyltransferase; sul1: sulphonamide resistance gene; aadA1: aminoglycoside
adenylyltransferase; mer: mercury resistance gene; qacE: quaternary ammonium compound-resistance
gene.
4. Bacterial Resistance to Multiple Antibiotics via Diverse Biochemical Mechanisms

Simultaneous resistance to several antimicrobial compounds in diverse pathogenic
bacterial populations has emerged as the major impediment for the successful treatment
of infectious diseases globally. Since bacteria develop such resistance very rapidly by
diverse resistance mechanisms, it has now become very difficult to treat even common
infectious diseases [93]. More specifically, our current standard treatment protocols fail to
produce a significant therapeutic response against those multidrug-resistant pathogenic
bacteria. Treatment failure due to the evolution of multidrug-resistant bacteria also leads to
a prolonged hospital stay with severe illness and can cause a higher risk of morbidity and
mortality. The occurrence of resistance to multiple drugs has been reported in many clinical
bacterial populations of both Gram-positive and Gram-negative origins. These populations
have been defined as multidrug-resistant organisms (MDROs) when they show in vitro
resistance to at least three or more antimicrobial classes, as described in the proceeding
sections. The most dangerous MDR bacteria belong to the Gram-negative family, such as P.
aeruginosa, E. coli, A. baumannii, and K. pneumoniae—a notorious producer of ESBLs, KPCs,
and MBLs (i.e., NDM). In Gram-positive bacteria, methicillin resistant S. aureus (MRSA),
vancomycin resistant enterococci (VRE), and extensively drug resistant M. tuberculosis (XDR)
are notable examples of the MDR bacteria [85,99,100]. Resistance to multiple antibiotics
(MDR) is mediated by a diverse array of mechanisms such as enzymatic mechanisms of
drug modification, target modification by mutations, enhanced efflux-pump expression
and altered membrane permeability [101].
4.1. Disabling Antibiotic Activities through Enzymatic Modification

Enzymatic modification of antibiotics has been reported in many clinical bacterial
populations. Such resistance mechanisms have commonly but widely been documented for
natural antibiotics, including aminoglycosides (i.e., kanamycin, amikacin, and tobramycin)
and beta-lactam antibiotics. In each case, certain enzymes can modify the chemical com-
position of the antibiotics, which in turn result in altered drug-target interactions [102].
Commonly observed aminoglycosides modifying enzymes are aminoglycoside acetyltrans-
ferase (AAC-3-II), aminoglycoside phosphorylase (APH-30 -I) and adenylate (nucleotidyl
transferases). For example, AAC-3-II can modify different aminoglycosides including
amikacin, gentamycin, and tobramycin. This enzyme is mostly carried on MGEs and
confer resistance to multiple antibiotics. It has been reported that multiple acetyltrans-
ferases encoding genes were harboured on class-1 integron in clinical P. aeruginosa, which
conferred resistance to many other classes of antibiotics, including carbapenems and sul-
fonamides [103]. Enzymatic inactivation of beta-lactam antibiotics is also common in
certain multidrug-resistant clinical bacteria. Genes located on plasmids mostly encode the
beta-lactam degrading enzymes, but chromosomal genes can also encode such enzymes.
Most clinically relevant beta-lactam hydrolyzing enzymes are beta-lactamase (first reported
in E. coli against penicillin), TEM beta-lactamase conferring resistance to multiple drugs is
commonly found in Gram-negative bacteria containing also multidrug resistant R plasmids,
and CTX-M beta-lactamase which was originally derived from the mobilization of chromo-
somal bla genes from Kluyvera spp. through mobile genetic elements. All these enzymes
belong to the ESBL (extended-spectrum beta-lactamase) class of enzymes [94,104,105].
Antibiotics 2021, 11, 40 11 of 23
4.2. Bypassing Antibiotic Interactions by Altering Target Sites

Bacteria develop antibiotic resistance as a result of mutations that make antibiotic
target sites less accessible. Mutational resistance has been observed in many multidrug-
resistant bacteria. For example, mutations in DNA gyrase (comprised of GyrA and GyrB)
and topoisomerase IV (comprised of ParC/GrlA and ParE/GrlB) give clinically relevant
resistance to fluoroquinolones [21,106]. Most of these mutations conferring resistance to
fluoroquinolone are in a region termed quinolone resistance determining region (QRDR)
of GyrA and ParC/GrlA. Interestingly, mutations in DNA gyrase occur in Gram-negative
bacteria, whereas mutations in topoisomerase IV appear in Gram-positive bacteria [93]. An-
other prominent example of resistance mediated by mutational target alteration is resistance
to rifampicin antibiotics. During tuberculosis infection, rifampicin in combination with
other drugs—including isoniazid, pyrazinamide, ethambutol, or streptomycin—remains
the first-line therapy [107,108]. A mutation in the rpoB gene, on the other hand, induces
a conformational shift in the RNA polymerase beta-subunit, which prevents rifampicin
from binding to its target. Mutations conferring resistance to aminoglycosides are also
common in many multidrug-resistant bacteria. For example, mutational alteration in small
ribosomal protein (S12) encoded by rpsL gene as well as mutation in rrs gene confers
resistance to streptomycin [51], which has been documented in M. tuberculosis [109].
4.3. Efflux Pumps Reduce Intracellular Antibiotic Concentration

Efflux systems are the significant contributors of natural antibiotic resistance in Gram-
negative microorganisms, which can effectively ship numerous antimicrobial agents out of
the cell [102]. Overexpression of efflux systems has been observed to be associated with
undeniable level protection from numerous clinically significant antibiotics. Efflux systems
in bacteria are grouped into two significant classes based on their substrate specificity:
(i) substrate explicit efflux system can just vehicle certain antibiotics (e.g., Tet efflux system
can just pump tetracycline antibiotics out of the cell); (ii) substrate unspecific efflux frame-
work can expel numerous antimicrobials—called MDR efflux systems. Chromosomal genes
typically encode such MDR efflux systems, however genes encoding MDR efflux systems
can also move onto plasmids, which can ultimately spread to different microbes. There
have been large numbers of such MDR efflux systems only found in the MDR bacterial
populaces, as mentioned in the preceding section.
4.4. Alteration of Membrane Permeability Prevents the Penetration of Antibiotics into

Bacterial Cells
Gram-negative bacteria are covered by two-membrane cell envelop (i.e., outer mem-
brane and inner membrane). The outer membrane is composed of the asymmetrical bilayer
of LPS and phospholipids. Porin proteins and uptake channels embedded into this outer
membrane bilayer regulate the entry of both hydrophobic and hydrophilic compounds into
the cell. However, during exposure to antibiotics, bacteria alter this permeability barrier
through modification of porin proteins. Porin proteins help bacteria to transport different
compounds in and out of the cell (Figure 3). Porin proteins are associated with multidrug
resistance. For example, OmpF and OprD are commonly occurring porin proteins found in
E. coli and P. aeruginosa, respectively. Both are involved in unspecific entry and exit points
for many different antibiotics and small chemical molecules. The membrane of P. aeruginosa
is about 10 to 80 times less permeable compared to E. coli. This restricted membrane
permeability provides built-in protections to P. aeruginosa against many different antibiotics
compared to E. coli. Overall, porin proteins embedded in the outer membrane are involved
in both acquired and adaptive resistance to multiple drugs. For example, imipenem and
meropenem are passed through this entry, and mutations can cause reduced levels of OprD
expression that confers resistance to these drugs. Furthermore, reduced porin expression
in Pseudomonas spp. and Acinetobacter spp., results in resistance to newer drugs, including
carbapenems and cephalosporins. Particularly, carbapenem mediated selective pressure
favours the emergence of mutations both in the genes encoding porin channels as well
Antibiotics 2021, 11, 40 12 of 23
as in the genes that regulate porin expression. Mutations in porin encoding genes in the
global K. pneumoniae lineages also contribute to the emergence of carbapenem resistance.
Furthermore, outer membrane porin protein such as Omp38 (or OpcP)—a homolog of
E. coli OmpF—mediates multidrug resistance in Burkholderia spp. There have been numer-
ous other bacteria where multidrug resistance through altered membrane permeability is
present [43,110].
5. Evolution of Antibiotic Resistance Is a Result of Natural Selection

Resistance to antibiotics is one of the best demonstrations of bacterial adaptation by
natural selection, a process in which a population adapts better to its environment over
time. Antimicrobial compounds exert selection pressure and thus drive the evolution
of resistance in bacterial populations. In the presence of antibiotic selection pressure,
mutations occur randomly through mismatch errors during DNA replication and repair.
This is how genetic variability—the raw material for the evolution of antibiotic resistance—
occurs in bacterial populations. At any time, a small number of individuals in a large
population carry de novo mutations or genes that are acquired by the HGT (more details
of the HGT will be discussed in the later section) from the surrounding environments. In
the presence of antibiotics, strains with resistance mutations are selected while others are
eradicated. Since resistant strains have the advantage to survive, they continue growing
and take over the population.
The rate of antibiotic resistance varies in types of antibiotics and bacterial species.
Exposure to a certain class of antibiotics results in hypermutator or mutator resistant geno-
types in the bacterial populations. This means that antibiotics exert an inhibitory effect on
bacterial physiology, which in response selects for mutator or hypermutator genotypes.
Several antibiotics belonging to different chemical classes can induce mutagenesis effect in
bacteria [23]. For example, a well-sought ROS (reactive oxygen species) oxidative damage
is induced by a sub-inhibitory concentration of a bactericidal antibiotic, as well documented
in E. coli [111], and a general stress response is also induced by such antibiotics [112]. Partic-
ularly, aminoglycosides trigger translational-stress-induced mutagenesis, fluoroquinolones
cause mutagenesis through DNA disruption, and beta-lactam classes of antibiotics can
induce SOS response in bacteria [113–115]. SOS response produces mutator phenotypes
by inducing error-prone DNA polymerase II, IV, and VI. A special case in point is cystic
fibrosis patients where hypermutator population of P. aeruginosa are generated by alteration
in the common genes which are involved in mismatch repair (MMR) system comprising
mutS, mutL, mutH, and uvrD; in other case, mutator alleles can also result from the presence
of antimutator genes, such as mutT, mutY, and mutM [116–118]. Another example is when
E. coli were confronted in rifampicin environment, a substantial proportion of the mutS–
populations fixed either one of the two transversion mutations, but the mutS+ strain was
fixed with a wide array of mutations [119]. This finding suggests that antibiotic resistance
evolves through genetic mutations in response to antibiotics and survival of the fittest
mutants occurs when natural selection acts on these mutations [114]. Mutation rate varies
from bacteria to bacteria due to the MMR system, as has been observed in E. coli and
S. enterica [120].
6. Horizontal Gene Transfer and Bacterial Recombination Promotes Evolutionary

Adaptation in Antibiotic Environments
HGT plays an important role in bacterial adaptation [121,122], particularly the evo-
lution of multidrug resistance. This process can act across species barriers [121–123],
provides genetic variation for natural selection acting on, and thus speeds up the adapta-
tion [124,125]. Through a single HGT, multiple traits can be transferred and integrated into
the recipient’s chromosomes, thus allow the hosts to expand their ecological niches, thrive
in harsh environments, or increase virulence [126,127]. Previously, HGT was proposed to
occur in only some bacteria. However, whole-genome sequencing in various species has
confirmed the pervasiveness and the dominant role of this mechanism over spontaneous
mutations in bacterial evolution [128,129]. It is estimated that at least 60% of prokaryotic
Antibiotics 2021, 11, 40 13 of 23
genes have undergone HGT [130]. Many different resistant determinants borne on MGEs
(i.e., plasmids, transposons, and integrons) are transferred and disseminated by HGT
within and between species. Furthermore, a recent study showed that HGT can act as a
directional force that allows neutral and mildly deleterious mutations conferring antibiotic
resistance to maintain in the populations at a very low frequency even without strong
selection [131]. At any given time, when selection pressure changes, strains with favorable
genotypes will rapidly spread.
Among various mechanisms of HGT, the acquisition of multidrug resistance through
recombination—i.e., natural transformation—could bring more evolutionary benefits to
the bacterial populations [132–134]. First, recombination can integrate single or multiple
DNA fragments into the recipient’s chromosomes [128,135]. This process increases genetic
variation in the populations and thus potentiates the adaptation to further environmen-
tal changes [131]. Second, recombination can prevent the accumulation of deleterious
mutations in populations during adaptation [136]. Since non-recombining populations
reproduce through vertical gene transfer—i.e., mother to daughter—the next generation
carry the same set of mutations as their mother. Mutations occur at any time and are often
more deleterious than beneficial [137]. Over time offspring maintain more deleterious
mutations leading to the deceleration of the adaptation. This process does not occur in
recombining populations because recombination can bring beneficial mutations into one
chromosome [136]. By shuffling available genetic variants in populations, recombination
also prevents clonal interference [133]. Due to the crucial advantages to the adaptation, bac-
terial recombination is considered as equivalence to sexual production in Eukaryote [138].
Despite the benefits, recombination is less likely considered in studies of antibiotic resis-
tance. Note that approximately 70% of the most dangerous human pathogens are naturally
competent [139]. It has been suggested that clinical A. baumannii acquired resistance to mul-
tiple drugs via transformation. Particularly, this bacterium acquired 45 different resistance
conferring genes from other species including E. coli, Pseudomonas spp., and Salmonella sp.
by transformation [140]. However, the dynamic of transferred genetic variants in these
recombining populations has not been mentioned.
7. Factors Affecting Evolutionary Dynamics of Resistance in Bacteria

Mutation supply rate determines the genetic variability in the infecting clonal pop-
ulations under antibiotic selective pressure, as mentioned earlier. Mutation supply rate
is determined by population size and rates of mutation and HGT in bacteria. However,
adaptive evolution of drug resistance, for example, the rate at which antibiotic resistance
will evolve and spread in the populations, is determined by several other factors, including
relative fitness of the resistant genotypes as the function of drug concentration, the strength
of selection pressure, clonal interference, population bottleneck or transmission bottleneck,
compensatory mutation, presence of epistasis, drug–drug interaction and coselection [141].
Relative fitness in the absence or presence of drugs is a key component in determining
the emergence and spread of resistant populations at a given mutation supply rate [78].
When selective pressure is altered, the frequency of resistant population or reversibility is
determined by the relative fitness cost. Consequently, fitness has been a key component in
determining and predicting the evolutionary adaptation of pathogen populations of both
laboratory and clinical origin [142–146].
Both natural and clinical bacterial populations face a wide array of selective pressures
in their surroundings, for example, in the environment (water and soil) or in certain
body compartments of humans or animals. Such concentration zones present in the
environment and in different niches of the body compartments favour the evolution and
amplification of drug resistant mutant subpopulations by reducing drug susceptibility in
the populations. According to the pharmacodynamic model, the selective zone for the
resistant subpopulations is called mutation selection window (MSW) [147]. The lower
boundary of this MSW promotes the elimination of the susceptible bacteria, which is often
referred to as minimum inhibitory concentration (MIC). According to traditional MSW
Antibiotics 2021, 11, 40 14 of 23
(i.e., drug concentration ranges between the MIC of susceptible and the MIC of resistant
bacterial populations—called mutant prevention concentration or the MPC), at high drug
concentration, the rate of resistance emergence is determined by the pre-existing mutation
in the populations. However, at low selective pressure, populations are enriched with many
small effect resistance mutations [148,149]. Consequently, it complicates the prediction
of mutational paths of antibiotic resistance due to the diversity of resistant mutants [150].
Low level of drug concentration, which is typically many folds below the MIC of the
susceptible bacteria, selects for resistance in the populations is called minimal selective
concentration (MSC). Therefore, sub-MIC selective window is important for the evolution
and maintenance of resistance in bacterial populations [151]. Furthermore, it has been
evident that strong selection pressure favours high level of cross-resistance (this can also
be termed as negative collateral sensitivity) to many antimicrobial classes, whereas under
low selection pressure populations enrich with weaker cross-resistance [152]. Together, this
suggests that the emergence and spread of resistant population is attributed to the strength
of selection pressure encompassing both the sub-MIC selective window and the traditional
MSW. Resistance to multiple drugs is the result of the concomitant presence of multiple
resistance conferring mutations in the individual clonal lineage of a given population [153].
When different beneficial mutations (i.e., a mutation is beneficial when it appears in an
individual bacterium in response to an antibiotic) arise in the populations, they compete
against each other, leading to the loss of most clones from the population. However,
clones with higher fitness outcompete the appearance of other clones with smaller fitness
effect [154]. This phenomenon is called clonal interference and has been well sought in
experimental resistant P. aeruginosa when evolved in the absence of antibiotic [155,156].
Epistasis—where the fitness effect of a mutation is influenced by another mutation
present in other genetic location—plays an important role in the evolution of antibiotic
resistance, multidrug resistance in particular [157]. Epistasis can occur between genes [158],
within a single gene encoding a single resistance protein [159,160], or between a chromoso-
mal gene and a gene encoded on a plasmid [161]. Several studies have identified pervasive
epistasis in bacterial adaptive evolution under a variety of conditions. For example, in two
studies, positive epistasis (when a double mutant has higher fitness than expected from the
sum of the costs of individual mutations in the absence of selection pressure) was reported
to the evolution of multidrug resistance [162,163]. Interestingly, this form of epistasis was
observed between a resistance mutation and a tolerance mutation in the presence of antibi-
otic selection pressure, and the authors have termed this as synergistic interaction [164].
In another study, Marta Lukačišinová and colleagues used a high throughput platform to
measure the evolvability of antibiotic resistance of hundreds of E. coli mutants from Keio
collection in a high control environment of antibiotic concentration [165]. This study not
only found the strong epitasis between resistance mutations but also identified genes that
control the evolution of antibiotic resistance through spontaneous mutations. Importantly,
strains that are more sensitive to antibiotics have a greater increase in resistance levels.
This phenomenon is also known as diminishing-returns epistasis: the effects of beneficial
mutations are stronger in less fit genetic backgrounds [166,167]. Although reduced use
or withdrawing of antibiotic use has been suggested to reverse antibiotic resistance [78],
epistasis plays a major role in determining the adaptive potential of resistant populations.
For example, in some form of epistasis—called reciprocal sign epistasis—the fitness of
multidrug resistant genotypes in the absence of drugs is greater than either of the singly
resistant genotypes. This means that the acquisition of additional new resistance deter-
minants (new resistance mutation or new resistance plasmid) can further accelerate the
fitness of the initial resistant genotype. Therapeutic options become narrower when this
form of epistasis arises in clinical bacterial pathogens.
Compensatory evolution is another important means of adaptive evolution of an-
tibiotic resistance which also involves epistasis. Antibiotic resistance is deleterious (on
bacterial fitness) in the absence of drug pressure. For example, in the absence of drug
pressure, resistance determinants often impose fitness costs in the form of reduced growth,
Antibiotics 2021, 11, 40 15 of 23
transmission, or virulence [168]. However, after certain generations intra or inter genomic
secondary mutations arise in the resistant bacterial population that restore the costs in-
curred by the initial drug specific resistance mutations. This phenomenon of adaptive
evolution has been reported in both in vivo and in vitro studies [142,169–171]. Compen-
satory mutations can be resistance mutations themselves, which can both compensate and
confer resistance to other antibiotics. For example, in the absence of antibiotic A, resistance
mutation incurs fitness cost, but after certain generations, this cost is compensated for
by a new secondary mutation. This secondary mutation simultaneously ameliorates the
fitness cost as well as confers resistance to a new antibiotic (i.e., antibiotic B). This form of
pleiotropic fitness effect of a compensatory mutation has recently been observed between
a streptomycin resistance mutation and a rifampicin resistance mutation in E. coli [163].
Moreover, it has been shown that compensatory mutations in multidrug resistance are
distinct from the single drug [172]. Therefore, restricted utilization of antibiotics might not
be an advocated solution to the antibiotic resistance crisis when compensatory mutations
have already evolved in the dominant resistant strains [86].
Drug interactions are important in determining bacterial evolutionary adaptation
to multiple drugs. Drug interactions have been classified into two types: physiological
interactions and evolutionary interactions [173]. During physiological interaction, two
antibiotics are used in combination, and they can produce either synergistic interaction,
antagonistic interaction, or they can suppress each other’s effect—called suppressive drug
interaction. Antibiotic drug interactions result from when the combined inhibitory effect
of two drugs is larger (called synergistic interaction which is more inhibitory) or smaller
(called antagonistic interaction, where higher MIC is needed to obtain the same level of
inhibition of synergistic drug pair) than expected based on an additive model. During
suppressive drug interaction, two drugs in combination produce a weaker effect, which is
less than the null additive expectation or weaker than the individual drug effect. It has been
reported that synergistic drug pairs, at a certain concentration threshold, potentiate the
evolution of resistance by extending the traditional mutant selection window towards the
sub-inhibitory concentration [174,175]. These studies have shown that during combination
therapy (i.e., drug A + drug B) certain drug specific resistance mutations arise first (i.e.,
resistance mutation evolves to drug A), which in turn diminishes the synergistic action
of that drug pair owing to the evolution of a drug specific resistance mutation at the first
place. Subsequently, this mutation confers an enhanced growth advantage against that
drug pair, which drives the acquisition of resistance mutation against the second drug
B, thus multiple resistance mutations evolve in the presence of a synergistic drug pair.
Synergistic drug pairs when used at low concentrations (i.e., below the MIC) can accelerate
resistance emergence. On the contrary, it has been suggested that with antagonistic drug
pair, certain mutations or mutations to drug A occur first which eventually breaks and
convert antagonistic interactions into synergistic. Thus, antagonistic drug pairs decelerate
resistance evolution to the second drug B. Evolutionary interactions are further classified
into two types: cross-resistance and collateral sensitivity [173]. Resistance mutations or
genes arising through either spontaneous mutation or HGT can simultaneously confer
resistance to another drugs (called cross-resistance) or become more sensitive to other
drugs (called ‘collaterally sensitive’) [176,177]. Cross-resistance is the function of the
evolutionary response to a single antibiotic. Therefore, cross-resistance is different from
the physiological interactions, which require drugs to be administered in combination.
Cross-resistance jeopardizes the efficacy of antibiotic treatments, while collateral sensitivity
provides the chance to prolong the effects of existing antibiotics as well as slow down the
rate of multidrug resistance [178,179]. Collateral sensitivity has been found to be pervasive,
however, its mechanisms and applications are not well understood [180]. For instance, the
evolutionary trade-off of resistance to ciprofloxacin P. aeruginosa is the increase in sensitivity
to two other drugs, piperacillin and tobramycin [181]. Another example is a long-term
experimental evolution in 24 days of E. coli in a morbidostat that automatically controls the
antibiotic treatments [182]. In this study, multidrug resistance was found to be prevented
Antibiotics 2021, 11, 40 16 of 23
by the trade-off of polymyxin B resistance. In both studies, the collateral sensitivity was
found to be unidirectional only. A more recent study provides additional details into the
factors that contribute to stabilizing the trade-off [183]. Importantly, the maintenance of
original pressure incurred by the first drug can maximize the hypersensitivity to the second
drug. Moreover, drug order is one of the important factors that can enhance or prevent
multidrug resistance.
Evolutionary dynamics of antibiotic resistance further rely on factors such as cos-
election and bottleneck. Coselection is a phenomenon where one resistance conferring
subunit (i.e., a particular resistance gene) is transferred with another subunit or another
gene (this process is often called hitchhiking), because the former gene (i.e., resistance
conferring gene) and the latter gene form the whole unit. Coselection is an important factor
in maintaining the resistance gene in bacterial population. For example, under given drug
pressure, a particular population will select for a resistance conferring mutation in a gene
by direct selection, or this gene will be selected with other genes (i.e., selection acts on a
gene that is co-linked with other genes). Coselection occurs within the genes localized on a
chromosome or between chromosomal genes and genes harboured on a plasmid, which are
closely associated with each other. A resistance conferring gene always incurs a fitness cost,
and coselection shields this resistance gene from purifying selection [184]. When sequential
drug perturbation occurs, coselection favours populations to select resistance for different
drugs in the co-linked genes. Thus, coselection favours the evolutionary maintenance of
genes conferring resistance to multiple drugs.
Population bottleneck is another important determinant for the adaptive evolution
of bacteria. For several reasons, this event is frequently observed in pathogenic bacteria,
including transmission from one host to another host, immune pressure, and antibiotics. In
the presence of antibiotic selective pressure, only resistant variants with higher fitness will
flourish in the population. However, the probability of the occurrence of this resistance
mutation in the subsequent generations is dependent on the population size or population
bottleneck. In the presence of a narrow transmission bottleneck, populations with higher
mutation rates (i.e., most commonly occurring mutants but they have low fitness) can
pass onto the next generation. With a wider transmission bottleneck, both commonly
occurring mutants with low fitness and rare mutants with higher fitness can transmit. In this
scenario, the fittest genotypes will increase their frequency and will subsequently displace
the genotypes with lower fitness. This competitive exclusion of low-fitness genotypes
can be minimized through the HGT and recombination. Depending on the size, the
population bottleneck also influences the occurrence of antibiotic resistance evolution
in both laboratory and clinical bacterial populations. Overall, examples of such event
can be drawn from several studies, for example multistep fluoroquinolone resistance in
E. coli [143,185] and rifampicin resistance in P. fluorescence [186].
8. Future Landscapes
Ever-increasing antibiotic resistance cases and a slowdown pipeline of novel antibiotics
have resulted in a shrinking toolkit to counter key clinically resistant bacteria. Thus, it is
crucial to elucidate the evolutionary mechanisms of origin, risk concerns and continued
supervision of antibiotic resistance to prolong the effective life of currently prevailing
antibiotics. Additionally, numerous bacterial species upon selection for antibiotic resistance
have hefty population density, shorter reproductive cycle, and robust selective pressures,
rendering them reasonable models for exploring more extensive, fundamental inquiries
with respect to fast adaptation against new selection pressures. This might incorporate
inquiries about the significance of de novo mutation and standing variations, the causes
underlying their variable prevalence in separate occasions, which adaptive choices are
available to an emerging bacterial population, etc. Identifying the probable sources of
antibiotic resistance before a clinical approval is adopted would expand the chances of
fruitful proactive antibiotic resistance management. If we can predict the evolutionary
trends of resistance of an antibiotic in bacterial species, it will be easy to customize antibiotic
Antibiotics 2021, 11, 40 17 of 23
dosing regimens and to achieve an extended duration of use. Statistical modelling of

medico-epidemiological data can generate a decent understanding of the relationship
between the rate of resistance evolution and geographical spread pattern. The rate of
resistance evolution in developing countries can be different from the developed countries
because the antibiotic consumption rate is much higher in developing countries viz. Brazil,
India, and China. Further research is required to understand the extent of concerns and
repercussions to cope with the threats of resistance. This review will provide a distinctive
reference for advancing healthcare settings, determining treatment regimes, and achieving
a clearer insight into the fluctuating epidemiology of resistant bacteria.
Author Contributions: C.M.H.: Conceptualization, writing original draft, reviewing, and editing;
A.N.T.N., D.D.: Writing, reviewing, and editing. All authors have read and agreed to the published
version of the manuscript.
Funding: C.M.H. was supported by the University of Queensland International Postgraduate Schol-
arship. D.D. at the University of Liverpool was funded by the CommonwealthSplit-site Scholarship,
Govt. of UK.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: We acknowledge an anonymous reviewer of an earlier version of this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Revisiting Antibiotic Resistance Mechanistic

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antibiotics

1 School of Biological Sciences, University of Queensland, Brisbane 4072, Australia

Received: 31 August 2021

Antibiotics 2021, 11, 40. https://doi.org/10.3390/antibiotics11010040 https://www.mdpi.com/journal/antibiotics

Antibiotics belong to different classes of chemicals—including those of biological,

Table 1. Action and common resistance mechanisms of major bacteriostatic antibiotics.

Bacteriostatic Candidates Mode of Action Mechanism of Resistance

Table 2. Action and common resistance mechanisms of major bactericidal antibiotics.

antibiotic exposure or horizontal gene transfer. Gram-positive bacteria are susceptible

3.1. Natural Defense Systems to Antibiotics

Bacteriophage Conjugation (3)

Resistance gene containing Donor cell

4. Bacterial Resistance to Multiple Antibiotics via Diverse Biochemical Mechanisms

4.1. Disabling Antibiotic Activities through Enzymatic Modification

4.2. Bypassing Antibiotic Interactions by Altering Target Sites

4.3. Efflux Pumps Reduce Intracellular Antibiotic Concentration

4.4. Alteration of Membrane Permeability Prevents the Penetration of Antibiotics into

5. Evolution of Antibiotic Resistance Is a Result of Natural Selection

6. Horizontal Gene Transfer and Bacterial Recombination Promotes Evolutionary

7. Factors Affecting Evolutionary Dynamics of Resistance in Bacteria

dosing regimens and to achieve an extended duration of use. Statistical modelling of

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