emergence and spread of amr · emergence and spread of amr date: jutkina, j., et al....

Emergence and spread

of AMR

Tim McAllisterPrincipal Research Scientist

Agriculture and Agri-Food Canada

Adjunct Professor

Faculty of Veterinary Science, Department of Production Animal Health

Cummings School of Medicine, Department of Physiology and Pharmacology

Email: [email protected]

Date: Sept 22, 2020

Learning Objectives

How bacteria became resistance to antibiotics?

How diverse the mechanism of resistance is?

Role of horizontal gene transfer in dissemination of resistance genes in ecosystem

Co-occurrence of resistance genes to antibiotics, biocides and metals on plasmids.

Emergence and spread of AMR Date:

Lecture Content

Antimicrobial resistance

Mechanisms of resistance

Selection of resistance isolate

Resistance before antibiotics

Dissemination of resistance genes

Horizontal gene transfer in different ecosystems


Bacterial warfare- the never ending battle


Lecture Title: Date:

3. Competitive exclusion

1. Competition for nutrients2. Direct antagonism

Approaches to bacterial warfare

Antimicrobial

resistance elements

from 4 human skeleton

Shigella flexneri isolate

resistant to penicillin and

erythromycin Available for

human use

First case of penicillin

resistance

Resistance before antibiotics


Discovery of

Penicillin

1927

1941

1947

1915

950–200 CE

Gut-microbiome of pre-

Columbian Andean mummy

from Cuzco, harbor resistance

genes

950-1170 AD

5k yrs.

presence of resistance

genes in Siberian

permafrost

presence of

vancomycin resistance

gene in permafrost

30k yrs.

Reference:Perry J, Waglechner N, Wright G. The Prehistory of Antibiotic Resistance.

Cold Spring Harb Perspect Med. 2016;6(6):a025197. Published 2016 Jun 1.

doi:10.1101/cshperspect.a025197

Forms of antimicrobial resistance

Acquired

➢ Mutation (endogenous and vertically transmission)

➢ Horizontal gene transfer (Conjugation, transformation and transduction)

Intrinsic

➢ Due to structural or functional trait allowing tolerance by all members of a group (species,

genus, etc)

✓ Inaccessibility of the drug into the bacterial cell (Gram-negative cell wall)

✓ Extrusion of the drug by chromosomally encoded active exporters (Pseudomonas)


Decreased uptake

Overexpression of

efflux pump

Enzymatic degradation

DNA

Target alteration

A B

Alterative enzyme

Mechanism of resistance

Pseudomonas aeruginosa can

produce pumps to get rid of

several different important

antibiotic drugs, including

fluoroquinolones, beta-lactams,

chloramphenicol, and

trimethoprim.

Klebsiella pneumoniae

produce enzymes called

carbapenemases, which

break down carbapenem

drugs and most other

beta-lactam drugs

Gram-negative bacteria have an

outer layer (membrane) that protects

them from their environment. These

bacteria can use this membrane to

selectively keep antibiotic drugs

from entering.

Staphylococcus

aureus can bypass

the drug effects of

trimethoprim

mutations in RNA polymerase

and DNA gyrase, resulting in

resistance to the rifamycins and

quinolones

Resistance to macrolide

involves modification of

their target site on the

ribosome.


Tetracycline resistance – a single example

of resistance mechanisms in bacteria

Active efflux: tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(I), tet(J), tet(Y),

tet(30), tet(31), tet(K), tet(L), tet(V), tet(Z), tetP(A), tcr3, otr(B), tet(33), tet(35),

tet(38), tet(39)

Ribosomal protection: tet(M), tet(O), tet(Q), tet(S), tet(T), tet(W), tetP(B), otr(A),

tet, tet(32), tet(36)

Enzymatic modification: tet(X), tet(34), tet(37)

Known - Unknown: tet(U), otr (C)

Unknown - Unkowns: ???


Overview of microbial drug resistance

Chromosome and

cytoplasm

RNA Polymerase

mutations

Rifamycin

DNA gyrase and

topoisomerase IV

mutations

Quinolones

Novobiocin

Ribosomal mutations

and modification

Aminoglycosides

Acquired metabolic by-

pass

Sulphonamides

Trimethroprim

Mupirocin

Reduction

Nitroimidazoles

Modifying

enzymes

Aminoglycosides

Chloramphenicol

Fosfomycin

Streptogramin A

Tetracyclines

Rifamycins

Inner

membraneCell wall

New or

mutated PBPs

Penicillin

Cephalosporins

Monobactams

Carbapenems

Beta-lactamases

Penicillin

Cephalosporin

Monobactams

Carbapenems

Acquired ligases

Vancomycin

Teicoplanin

Outer membrane

(Gram –ve)

Permeability and porin deficiency

Penicillins

Cephalosporins

Monobactams

Carbapenems

Quinolones

Aminoglycosides

Tetracyclines

Membrane pumps

Tetracyclines

Fusidic acid

Quinolones

Bacitracin

Chloramphenicol

Lincosamides

Betalactams

Aminoglycosides

Macrolides

Antiseptics


One DrugSeveral

Mechanisms

Many

Bacteria

Variable

phenotype

Variable

clinical

importance

Mode of

acquisition

Mechanism Bacterial species

None Reduced

permeability

Pseudomonas spp.

Conjugation Enzymatic

inactivation

Enterobacteriaceae

Staphylococci

Unknown Target replacement Staphylococci

Transformation Target modification Streptococci

Ex: Beta-lactam

resistance


Selection of resistance

The origin of species by means of natural selection,

1859 Charles Darwin

Variation within species occurs randomly and that survival or extinction of

each organism is determined by that organism’s ability to adapt to its

environment


”Snowball rolling downhill effect”


Time

An

tib

ioti

cs s

uscep

tib

ilit

y

Dissemination of resistance genes

Vertical transmission Horizontal transmission

The mobilization of ARGs on

plasmids, transposons, and

integrons has enabled resistance

genes to spread horizontally

across strains and species,

greatly facilitating the

development of resistance in

numerous pathogens.



• In animal farming

• In community

• In healthcare facilities

• Through travel

https://www.youtube.com/watch?v=4KMWun3Og8I

Drugs makes bugs (3min video)

https://www.youtube.com/watch?v=4KMWun3Og8I

Horizontal gene transfer in ecosystemsSoil ecosystems harbor an extremely

broad diversity of microbiota reflecting

plant type, soil type, soil management

regime, and other factors

More than 70% of the earth

surface is covered with water,

and the World Ocean is one of

the principal components

forming the climate and

biosphere of the Earth.

biofilm communities are widespread in

many natural ecosystems. Majority of

microbiota found in natural, clinical, and

industrial settings persist in association

with surfaces and not in the planktonic

state

Conditions in the gut can be considered as very favorable for

HGT. Because of continuous inflow of nutrients, the

population densities are extremely high and thus conducive

to the HGT mechanisms requiring intimate cell-to-cell contact

such as conjugation.

Ref: Aminov RI (2011) Horizontal gene

exchange in environmental microbiota.

Front. Microbio. 2:158. doi:

10.3389/fmicb.2011.00158

HGT in soil ecosystem

The microbiota of soil, especially of manure-fertilized soil, harbors a wide variety

of MGEs enabling extensive HGT within and among microbial ecosystems.

Clinically relevant class 1 integrons are also introduced into soil via manure

application.

A novel plasmid type with low G + C content was captured from manure-treated

soil microbiota by conjugation to E. coli recipient.

The species of Acinetobacter are the putative hosts for low G + C plasmids in soil

ecosystem.

Similar to the plasmid-mediated HGT, the diversity, and dynamics of

bacteriophages is also mostly confined to the nutritionally rich hot spots such as

the rhizosphere.

The possibility of natural genetic transformation of B. subtilis and Pseudomonas

stutzeri has been demonstrated in a soil/sediment model system


HGT in aquatic ecosystem

Antibiotic resistance phenotype and the presence of plasmids in marine bacteria tend to

correlate with the degree of pollution, especially by toxic chemical waste.

Resistance phenotype was more frequently encountered in bacteria from seawater samples

collected offshore than for those collected near shore.

Plasmids with an enormous size range are also widespread in the Roseobacter clade (25%

of the total marine bacterial community) and thus play a global role in this ecosystem


HGT in gut ecosystem

The intestinal microbial ecosystem is extremely enriched by MGEs thus making it the

arena of a potentially extensive gene exchange.

The discovery of ICEs among the representatives of Bacteriodetes, the major bacterial

phylum in the mammalian gut.

Indeed, the taxonomically different representatives of gut microbiota may share the pool of

closely related antimicrobial resistance genes.

The occurrence of conjugal plasmid transfer and transduction was also observed in the

flea, house fly gut and ciliate's food vacuole.


HGT in biofilm ecosystem

Biofilms are the matrix-enclosed aggregates of microbiota, attached to each other

and to biological or non-biological surfaces

Biofilm formation is linked to the pathology of many infectious diseases, and

biofilms are notoriously difficult to eradicate because of the increased level of

resistance to antibiotics.

They are the hot spots for HGT because they provide high population densities

and close proximity of cells, cells in biofilms are metabolically active, and the

microbiota is protected against harsh environment, predators, and immune

surveillance by the extracellular matrix in which the cells are encapsulated.


Conjugal plasmid transfer that is implemented through the synthesis of pili and

type IV secretion system contributes to the intimate cell-to-cell contact, thus

facilitating the formation and growth of biofilms.

The presence of prophages in the host’s genome may have a modulatory effect on

biofilm formation.

A partial phage-mediated lysis of a proportion of microbiota in biofilm, due to

spontaneous phage induction, may provide the eDNA supply, thus contributing to

biofilm formation and maintenance by the remaining bacterial populations.


Pollution linked to AMR

Heavy metals in environmental reservoirs may contribute to

selecting antibiotic-resistant strains

Pseudomonas aeruginosa and Escherichia coli - co-occurrence of

resistance to several heavy metals and antibiotic classes.

Cross-resistance – One resistance system confer resistance to

both.

Co-resistance – Resistance gene co-located on same genetic

element

Co-regulation – Transcriptional and translational regulation by

same regulator in response to stress


Reference: Nguyen, C.C., Hugie, C.N., Kile, M.L. et al. Association between

heavy metals and antibiotic-resistant human pathogens in environmental

reservoirs: A review. Front. Environ. Sci. Eng. 13, 46 (2019).

https://doi.org/10.1007/s11783-019-1129-0

Zinc, a common and toxic metal known to

co-select for antibiotic resistance with

industrial and agricultural activities

Bacterial isolates from the sediment were

used to test for patterns of cross resistance

between zinc and cefotaxime, oxacillin and

trimethoprim.

Significant increase in tolerance to these

antibiotics in isolates that exhibit resistance

to zinc.


Reference: Dickinson, Andrew William, et al. "Heavy metal pollution

and co-selection for antibiotic resistance: A microbial palaeontology

approach." Environment international 132 (2019): 105117.

HGT and heavy metals


Heavy metal Cu promotes the plasmid

RP4 mediated conjugative transfer

environmentally by cell damage effect,

facilitating the transmission of ARGs.

Reference: Wang, Qing, et al. "Heavy metal copper accelerates the conjugative transfer of antibiotic

resistance genes in freshwater microcosms." Science of The Total Environment 717 (2020): 137055.

HGT and biocide

The global Biocide consumption is estimated

at $ 6.6 billion

The use of biocides might lead to increased

resistance to antimicrobial agents used for

treatment of humans and animals, through

increasing mutation rates in bacteria or

increasing horizontal gene transfer.

Ethanol, chlorine, chloramine, H2O2 and,

nano-titanium dioxide (TiO2) can promote

conjugal transfer of resistance plasmids.

Triclosan and chlorhexidine induced

horizontal gene transfer at low

concentrations.

Emergence and spread of AMR Date: Jutkina, J., et al. "Antibiotics and common antibacterial biocides stimulate

horizontal transfer of resistance at low concentrations." Science of the total

Environment 616 (2018): 172-178.

Co-occurrence of resistance genes to

antibiotics, biocides and metals on plasmids.


The network shows most

frequently observed co-occurrence

of resistance genes to antibiotics,

biocides and metals on plasmids.

Connection between

metal/antibiotic resistance genes

and markers of MGEs

Reference: Pal, Chandan, et al. "Metal resistance and its association with antibiotic resistance." Advances in microbial physiology.

Vol. 70. Academic Press, 2017. 261-313.

Additional Resources

https://www.nytimes.com/video/health/100000005932983/bacteria-antibiotics-war.html

Forsberg KJ, Reyes A, Wang B, Selleck EM, Sommer MO, Dantas G. The shared

antibiotic resistome of soil bacteria and human pathogens. Science.

2012;337(6098):1107-1111. doi:10.1126/science.1220761

Granato, Elisa T., Thomas A. Meiller-Legrand, and Kevin R. Foster. "The evolution and

ecology of bacterial warfare." Current biology 29.11 (2019): R521-R537.

Bush, K. Past and present prespective on beta-lactamases. Antimicrobial agents and

chemotherapy. https://doi.org/10.1128/AAC.01076-18.


https://www.nytimes.com/video/health/100000005932983/bacteria-antibiotics-war.html

Summary

Bacteria have different mechanisms of antibiotic resistance

There may be different ways to confer resistance to one single

antibiotic or vice versa (cross resistance)

Different ecosystems facilitate the dissemination of resistance gene

among different and similar species.

Resistance to heavy metals and biocide are linked to antibiotic

resistance.


Questions


Break!

10 minutes break

After Break

Discussion session


Discussion Question(s)


What are some of the factors associated with spread of

antimicrobial resistance from livestock production systems

that should be considered when trying to gage potential

risks to human health?

Thank You!

Email: [email protected]

Twitter: timmcallister7


Co- Chair VTEC 2021 Web Site: https://vtec2021.org/ https://www.facebook.com/vtec2021 https://twitter.com/vtec2021

mailto:[email protected]

emergence and spread of amr · emergence and spread of amr date: jutkina, j., et al....

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