UNIVERSITATEA DE TIINE AGRICOLE I

MEDICIN VETERINAR CLUJ NAPOCA

ANTIBIOTIC RESISTANCE
an overview of
horizontal gene transfer mechanisms

S. GURANDA, ELENA MARIAN, MARINA SPNU

 1. INTRODUCTION
Infections - leading cause of death (beginning of the
20th century)

Penicillin (Alexander Fleming, 1929)

Sulpha drugs (Gerhard Domagk, 1932)

Golden era of antibiotics which peaked

between 1940 and 1970

Survival of the fittest no absolute control over infections!

Infectious diseases second-leading cause of death worldwide

(>13 million deaths/year)

Post-antibiotic era antimicrobial resistance global report (April

2014)
 1. INTRODUCTION
Antibiotic microbial resistance (AMR) a world-wide problem not
only in human, but also in veterinary medicine

Bacteria rapidly growing organisms

Antibiotics extensive & irrational use

Constant selective pressure - results in increased antibiotic

resistance (due to genetic mutations and horizontal gene transfer)

Donors and recipients of horizontal gene

transfer (HGT) might belong to different
bacterial species and genera, located in
various ecosystems
One planet, one medicine, one health
 1. INTRODUCTION
Antibiotics what are they?

Traditional paradigm
antibiotics seen as merely
weapons for fighting bacteria

Paradigm shift
antibiotics are also seen as

signaling molecules that may regulate the homeostasis of

microbial communities
sources of nutrition to microorganisms
2. Antibiotics efficacy and inefficacy
Antibiotics antimicrobial agents used specifically against bacteria
inhibit the growth / kill the bacteria, but ineffective against viruses

Mechanisms of action of antibiotics

(adapted from Mrculescu, 2008)
2. Antibiotics efficacy and inefficacy
low molecular compounds in natural environment - antibiotic effect
in high concentrations, but unlikely to reach such concentrations. Their
primary role is mostly not cross-species warfare, but cell to cell
communication.

Quorum sensing a phenomenon that limits certain behaviors to

occurring only above a certain population density (a system of stimulus
and response, used to coordinate gene expression according to the
density of local bacterial population (signaling molecules -
autoinducers or pheromones)
2. Antibiotics efficacy and inefficacy

History of antibiotic discovery and concomitant development of antibiotic

resistance (adapted from Davies et al., 2010)
2. Antibiotics efficacy and inefficacy

http://slatestarcodex.com/2014/07/24/some-antibiotic-stagnation/
 3. Resistance to antimicrobials
Microbial drug resistance - Paul Ehrlich (1907)
arsenical chemotherapy against trypanosomiasis

Resistance
Intrinsic resistance
Acquired resistance

3.1. Intrinsic resistance

natural insusceptibility of an organism to an antimicrobial
passive resistance a consequence of general adaptive processes
the same mechanisms could be involved in resistance to different
types of antibiotics.
the determinant of bacterial response to an antibiotic is the presence or
absence of the target for the action of the drug (4, 5, 23).
 3. Resistance to antimicrobials

3.2. Acquired resistance

the major mechanism involved in resistance
active resistance the result of a specific evolutionary pressure, in the
presence of an antimicrobial, in order to develop at least one
mechanism against that particular antimicrobial, which could be useful
also toward an entire class of antimicrobials
loss of sensitivity toward an antimicrobial happens in the presence of
clinically achievable concentrations of a drug.
the antibiotic exerts a selective pressure which favors the resistant
microorganisms and destroys the susceptible ones
bacteria may acquire resistance by
a mutation which can be passed vertically by selection to
daughter cells or to other bacteria of different species or genera
horizontal transfer of genes (HGT) responsible for coding the
resistance mechanisms.
 3. Resistance to antimicrobials
3.3 Antibiotic resistance mechanisms and emergence factors
five major mechanisms responsible for it:
(a) enzymatic degradation or modification of agent

(b) decreased uptake or accumulation of agent

(c) altered target

(d) circumvention of consequences of agent

(e) uncoupling of agent-target interactions or any combination of the

above mentioned
emergence of AR is due to four main factors:
(i) emergence of new genes (i.e. MRSA, VRE, VISA/GISA)

(ii) spread of old genes to new hosts (i.e. PRGC, GRSA)

(iii) mutations of old genes resulting in more potent resistance (i.e.

ESBLs)
(iv) emergence of intrinsically resistant opportunistic bacteria (i.e.
Stenatrophomonas maltophilia)
 3. Resistance to antimicrobials
Drug-resistant organisms genetically differ from the wild types,
which led to the idea that spontaneous mutations must be involved

Spontaneous mutations could arise in four ways:

damage to the genome caused by adverse environmental factors
(ionization radiation, chemical mutagens etc.)
base-pairing errors during DNA replication

frameshift mutations caused by the deletion of segments of DNA

which frequently occur at short DNA repeat sequences
frameshift mutations caused by the intragenic insertion of
mobilizable genetic material (i.e. transposons, which corrupt the
correct flow of information from the wild-type genome

The bacterial mechanisms for acquiring AR:

the modification of existing genetic material

the acquisition of new genetic material from an external source

 3. Resistance to antimicrobials
3.4 Horizontal gene transfer
a remarkable ability to mobilize genes in both chromosomal and
plasmid DNA and to transfer and exchange genetic information

AR might be transferred from resistant to sensitive bacteria by

horizontal gene transfer (HGT)
HGT unidirectional gene exchange
(donor to recipient) between cellular
organisms close or distantly related or
between a replication competent virus and
a bacteria
the genetic material a fraction of the genome (DNA or RNA),
most usually a gene or part of a gene (may or may not include
coding and/or non-coding sequences)

Result of HGT a stable integration into the recipients genome

(the transferred gene will be perpetuated in the offspring of the
recipient organism vertical gene transfer (VGT).
 3. Resistance to antimicrobials
3.5 Mechanisms of HGT
transformation, conjugation and transduction
 3. Resistance to antimicrobials
3.5.1 Transformation
Natural transformation uptake of free
macromolecular DNA by competent
bacteria (efficient absorption and
integration of DNA into their genome).

DNA can be used as a nutrient source, for DNA repair or for genetic
innovation.

DNA fragment is integrated into the genome either by recombination

or by forming an autonomously replicating element.

In laboratory frequency of transformation of genetic markers can

be as high as 10-3 (one cell in every thousand integrates a particular
gene)
 3. Resistance to antimicrobials
3.5.2 Transduction
Transduction transfer of a non-viral
DNA from an infected host bacterium
to a new host via infectious or non-
infectious viruses

Part of the host DNA is mistakenly packaged into the empty phage
head (its genome contains up to 10% of the bacterial DNA)

The phage might carry bacterial genes for antibiotic resistance (AR)
and is released by lysis, extrusion or budding (asexual reproduction)

The phage delivers the DNA carried in the capsid.

The integration of injected bacterial DNA into the recipient genome

requires the presence of homologous DNA sequences or specialized
integrases
 3. Resistance to antimicrobials
3.5.3 Conjugation
main mechanism for the spread of
antibiotic resistance

mediated by resistance plasmids

(R-plasmids = circular DNA in both
closed and nicked forms

donor cells (R+) have the ability to produce sex pili at their surface to
come in contact with sensitive receptor (R-) cells (mating pairs)

receptor cells (R-) get a copy of the R-plasmid and becomes drug-
resistant
most conjugative plasmids have specific systems ensuring that a
similar element is not yet contained by the recipient cell

Gram-negative and Gram-positive bacteria use a diversity of

conjugative systems
 3. Resistance to antimicrobials
3.5.4 Other mechanisms of HGT

vesicle-mediated translocation Gram-negative bacteria (i.e.

Neisseria gonorrheae, E. coli, Pseudomonas aeruginosa) can bud off
vesicle structures containing genetic material that can later fuse with
another bacterium, giving to the receptor the donors AR capacities

pseudovirus (virus-like) particles trap random fragments of the

genome, infect target cells and transfer them to another bacterium
(express effector or reporter molecules).

might be formed by bacteria that have genes that encode

proteins capable of forming such gene transfer agents
 3. Resistance to antimicrobials
3.5.5 Factors that affect the transfer of MGEs

Soil, aquatic environments and phytosphere

conditions for colonization, mixing and bacterial activity

limited resources for microbial growth that can severely limit

bacterial population densities and activity, which, in turn, affects
the efficiency of HGT mechanisms.

HGT
increases genetic diversity

excessively genetic baggage and the import of deleterious

genes.

Organisms
The efficacy of barriers (physical, biochemical and genetic) to restrict the
frequency of HGT increases proportionally with genetic distance
between donor and receptor (the frequency of HGT is much greater
within species than between unrelated or distantly related species)
 4. Conclusions

Bacterial genomes are open to genetic information exchange, thus

spreading antibiotic resistance

Further investigate selection in natural microbial habitats of bacteria

possessing lateral transferred genetic material and to develop new and
more efficient strategies to reduce the impact of antibiotic resistance for
animals and humans, in the effort to make the paradigm of one world,
one health, one medicine become a priority for our society.

Louis Pasteur: If we could intervene in the antagonism observed

between some bacteria, it would offer perhaps the greatest hopes for
therapeutics
Thank you for your attention!