Dynamics of Resistance Evolution

Lourens Robberts, PhD. 2007

How does resistance become a problem for patients?

Ü Acquisition of already resistant pathogen from environment

Ü Selection of already resistant strains from within patient (enrichment)

Ü  Imposing antimicrobial pressure on wild-type (sensitive) pathogen to create the necessary conditions for evolution of resistance mutations

Ü  Imposing pressure on wild-type pathogen to acquire and maintain exogenous resistance genes

Bacterial genetics:

Conjugative transfer Transformation,

Mutation

NEW BACTERIAL

RESISTANCE

Human investment:

R & D

NEW ANTIBIOTIC

Ü  Phenotype (observable resistance mechanisms) derive from the genotype

Ü  How does a new genotype (new genes/altered genes) arise that enables defence against antimicrobials?

Survival of GENES

Epidemic spread of clones viz GENES

Development of a mutation in the gene

Spread of the gene among hosts (bacteria), and spread of hosts with new gene

Environmental pressure on the host

Mutations of pre-existing genetic determinants

Type of mutation Resistance phenotype

Structural Streptomycin Rifampicin Fluoroquinolones Sulfonamides Trimethoprim

Regulatory Aminoglycosides (aarA) β-lactamases (AmpC) Chloramphenicol Fluoroquinolones Tetracyclines (marA) Imipenem (OMP)

Acquisition of foreign DNA Phenotype Acquired genes

Aminoglycosides Aminoglycoside-modifying enzymes

β-lactams β-lactamase genes

Chloramphenicol CAT genes

Erythromycin/clindamycin Methylase/MLSB genes

Methicillin mecA gene

Penicillin PBP genes, β-lactamase

Sulfa DHPS gene

Trimethoprim DHFR gene

Tetracyclines Tet resistance genes

Vancomycin Abnormal ligase and accessory genes

Mutations of acquired genes

Type of mutation Resulting gene / phenotype

Structural ESBL

Regulatory Mec (methicillin resistance) ESBL

MUTATION

ENVIRONMENT

ADAPT

EVOLUTION

MAINTENANCE

STABILITY

FAITHFUL

GENERATION

UV exposure

Ionizing radiation

Chemical exposure & Cellular metabolism

Oxidative deamination

Fidelity

Natural causes of point mutations

STABILITY Ü Maintenance of genome stability

Ü Faithful copying over many generations

Ü  Proof-reading (exonuclease activity of Pol III) Ü  Mismatch repair systems

Mutations are random with respect to their effect on the fitness of the organism (host)

Translation and the Genetic Code

Ü  61 sense codons and 3 stop codons

Ü  61 sense codons and 20 primary aa’s, 18/20 aa’s are encoded by >1 codon = degenerate code.

Mutation Ü  Substitution mutations

Ü  Transitions (purine – purine eg A → G) Ü  Transversions (pyrimidine → purine eg A → C) Ü  Synonamous – silent mutations – no aa change (degenerate

code)

Ü  Nonsynonamous – aa replacement Ü Missense Ü Nonsense

Ü  30% of all 3rd position changes are nonsynonamnous Ü  100% of all 2nd position changes are nonsynonamous Ü  96% of all 1st position changes are nonsynonamous

Insertions / deletions

Recombination

Ü Homologous recombination Ü Site specific recombination

1.  Unequal crossing over 2.  Intrastrand deletion

n  Site-specific rec when a repeated sequence pairs with another in the same orientation on the same chromatid

n  Excision of a transposable element can involve recombination between direct repeats, 5 – 9 bp, flanking the element

3.  Slipped-strand mispairing

Amino acid Venn diagram

Amino acid Series of amino acids – protein secondary structure

Tertiary protein structure

Surfaces, pockets, binding domains, conformational changes

Mutations of preexisting genetic determinants

Type of mutation Resistance phenotype

Structural Streptomycin Rifampicin Fluoroquinolones Sulfonamides Trimethoprim

Regulatory Aminoglycosides (aarA) β-lactamases (AmpC) Chloramphenicol, Fluoroquinolones, tetracyclines (marA) Imipenem (OMP)

NH

NNH

NNH2

O

OP

OP

OO

O

O

O

NH2 COO-

NH

NNH

N

O

NH2

CH2

NH

COO-

NH

NNH

NNH2

O

NH

NH

O

-O2CCOO-

NH

NH

NH

N

O

NH2

NH

NH

O

-O2C COO-

Dihydropterin pyrophosphate (DHPPP)

p-Aminobenzoate (pABA)

7, 8-Dihydropteroate (DHP)

Dihydrofolate (DHF)

Pyrophosphate

Dihydropteroate synthase (DHPS)

ATP + Glutamate

NADP

NADPH

Dihydrofolate reductase (DHFR)

Sulphonamide

+ DHP-Sulpha

Dihydrofolate synthase (DHFS)

Tetrahydrofolate (THF)

Trimethoprim

Wild type

Mutant

Pneumocystis jirovecii DHPS

Mutations of preexisting genetic determinants

Type of mutation Resistance phenotype

Structural Streptomycin Rifampicin Fluoroquinolones Sulfonamides Trimethoprim

Regulatory Aminoglycosides (aarA) β-lactamases (AmpC) Chloramphenicol, Fluoroquinolones, tetracyclines (marA) Imipenem (OMP)

Group A

TEM & SHV

Pen Cef ’s

Penems

Inhibitor Sensitive

Group C

AmpC

Cef ’s Oxa

Inhibitor Resistant

Group D

OXA

Pen esp Oxa

Inhibitor

S / R

Group B

IMP & VIM

Penems

Inhibitor Resistant

Active site Serine Active site Zn

(metallo)

β-lactamase classification

http://www.psc.edu/science/2006/enzyme

Gram-positive

•  Group A

Components

B. licheniformis

1.  blaR1

2.  blaR2

3.  blaI

4.  blaP

Gram-negative

•  Group C

Components

C. fruendii

1.  ampC

2.  ampR

3.  ampD

4.  ampG

Bennett, PM. Antimicrob Agent Chemother 1993;37(2).

Gregory, PD. Mol Microbiol 1997;24(5).

Jacobs, C. Science 1997;278(5344).

Inducible Gram-positive β-lactamase

blaR1 blaI O blaP

blaP blaI

blaR1

blaP blaP

blaP

blaR2

blaR2

β-lactams

blaR1 blaI O blaP

blaP blaI

blaR1

blaR2

blaR2

Inducible Gram-positive β-lactamase

blaR1 blaI O blaP

blaP blaI

blaR1

blaR2

blaR2

Noninducible basal-level expression of blaP

Inducible Gram-positive β-lactamase

blaR1 blaI O blaP

blaP blaI

blaR1

blaP blaP

blaP

blaR2

blaR2

Constitutive high-level expression of blaP

Inducible Gram-positive β-lactamase

Gram-negative

•  The inducible β-lactamases are exclusively chromosomal genes

•  AmpC – extended phylogenetically related family, some members are no longer inducible e.g. β-lactamases of E. coli, Shigella and Salmonella spp.

Inducible Gram-negative ampC β-lactamase

Inducible Gram-negative ampC β-lactamase

Inducible Gram-negative ampC β-lactamase

ampD Null mutant

Derepressed

Constitutive hyperproducer

ampD Hyperproducer

More sensitive to inducer

3 – 4X expression

ampR Non-inducible

2 – 3X expression

Acquisition of foreign DNA

Phenotype Acquired genes

Aminoglycosides Aminoglycoside-modifying enzymes

β-lactams β-lactamase genes

Chloramphenicol CAT genes

Erythromycin/clindamycin Methylase/MLSB genes

Methicillin mecA gene

Penicillin PBP genes, β-lactamase

Sulfa DHPS gene

Trimethoprim DHFR gene

Tetracyclines Tet resistance genes

Vancomycin Abnormal ligase and accessory genes

Bacteria have inhabited the earth for > 3.5 billion years, competing for survival, and evolving chemical defenses against

rival species – antibiotics.

Some clinically important antibiotics

Antibiotic Producer organism Activity Site or mode of action

Penicillin Penicillium chrysogenum Gram-positive bacteria Wall synthesis

Cephalosporin Cephalosporium acremonium Broad spectrum Wall synthesis

Griseofulvin Penicillium griseofulvum Dermatophytic fungi Microtubules

Bacitracin Bacillus subtilis Gram-positive bacteria Wall synthesis

Polymyxin B Bacillus polymyxa Gram-negative bacteria Cell membrane

Amphotericin B Streptomyces nodosus Fungi Cell membrane

Erythromycin Streptomyces erythreus Gram-positive bacteria Protein synthesis

Neomycin Streptomyces fradiae Broad spectrum Protein synthesis

Streptomycin Streptomyces griseus Gram-negative bacteria Protein synthesis

Tetracycline Streptomyces rimosus Broad spectrum Protein synthesis

Vancomycin Streptomyces orientalis Gram-positive bacteria Protein synthesis

Gentamicin Micromonospora purpurea Broad spectrum Protein synthesis

Rifamycin Streptomyces mediterranei Tuberculosis Protein synthesis

The other side of the coin however:

Many species of pro- and eukaryotes (especially fungi) have equally evolved counter measures against antibiotics for an equal amount of time.

Now an environmental library of resistance genes exist.

Normal microbiota

Artificial environments e.g. indwelling medical devices

Ü Cattle 104 – 110 million

Ü Chickens 7.5 – 8.6 billion

Ü Turkey 275 – 292 million

Ü Swine 60 – 92 million

Ü Antibiotics used: 9.3 million Kg / year

Ü Meat producing animals excrete 1400 billion Kg waste / year

Agriculture & Veterinary

Sarmah, AK. Chemosphere. 2006;65.

Central lending library: Mechanisms of acquiring foreign DNA

(Horizontal Gene Transfer)

Genetic mechanisms of antibiotic resistance acquisition among common pathogenic bacteria

Mutation Natural transformation Conjugative transfer

All bacteria M. Tuberculosis

Acinetobacter Enterococcus Helicobacter Haemophilus Neisseria Staphylococcus Streptococcus

Enterobacteriacaea* Acinetobacter Campylobacter Bacteroides Clostridia Enterococcus Haemophilus Helicobacter Mycoplasma Listeria Neisseria Pseudomonas Staphylococcus Streptococcus Vibrio Yersinia

* Enterobacter, E. coli, Klebsiella, Proteus, Salmonella, Shigella, Serratia

Hospitals: Convenient ecosystem for gene transfer

Ü Many patients Ü Continuous change Ü Reservoirs Ü Selective antibiotic

pressure

Genetic pool and HGT “selfish gene”

Ü  Ability to take up DNA Ü  Willingness to deliver

DNA Ü  In proximity at the same

time Ü  Encountering free DNA

circulating in the environment

Bacteriophages are bacterial viruses

Ü  Major contributor to the evolution of bacteria

Ü  DNA of phage origin often comprize 10 – 20% of bacterial genomes

Ü  2/3 of gamma proteobacteria harbor intact / remnant bacteriophage genomes

Ü  Ubiquitous in GIT (107/g), marine and soil systems, and sewage

Transduction

Ü  Process by which bacteria take up naked (free) DNA from the environment

Ü  Restrictions apply (restriction modification system)

Ü  S. pneumoniae, viridans streptococci, H. influenzae, N. gonorrhoea,

N. meningitidis

Transformation

Homologous recombination

Fate of incoming foreign DNA

S. pneumoniae PBP2b

Wild-type

United Kingdom 1987

Chech Republic 1987

Papua New Guinea 1970

Kenya 1992

South Africa 1990

Papua New Guinea 1970 20%

4%

30%

21%

S. pneumoniae

S. mitis

S. oralis

Strep?

Strep?

Dowson, CG. Trends Microbiol. 1994;361.

Dessen, A. J Biol Chem. 2001;276.

Structural comparison between Sp328 and R6 PBP2x*

Figure shows superposition of PBP2x* from Sp328 (green) and from the penicillin-sensitive R6 strain (blue). Most C chain divergences occur at the level of the N-terminal domain, which is much more stable and the 360–394 loop region (red), flexible in the penicillin-resistant molecule.

Dessen, A. J Biol Chem. 2001;276

Drug-sensitive and -resistant active sites

A, active site of PBP2x* from penicillin-sensitive strain R6 (1QME.pdb). The three crucial motifs for enzymatic activity are represented by Ser337 (SXXK), Ser395 (SXN), and Lys547 (K(S/T)G). Thr338 is at the N-terminal end of 2, and Asn514 points away from 4, which harbors Ser389. B, active site of PBP2x* from penicillin-resistant strain Sp328. Although all three motifs are represented, the SXN loop is clearly displaced, probably the result of a steric clash between Leu389 and His514, which points into where 4 should be located. Ser347 not only adds a polar character to the region but also makes contact with Thr352, a residue present in the loop that follows 2 (not shown for clarity).

Conjugation

Ü  Method by which bacterial cells come into contact with each other to exchange genetic material

Ü  Machinery required for conjugation (pilus and transfer) encodes by a plasmid in the donor

Plasmids are extra-chromosomal circular DNA. Encodes accessory functions including antimicrobial resistance, carbohydrate fermentation, bacteriocins, toxins, adhesive and colonization factors, conjugation

Natural history of emergence of resistance to β-lactams and aminoglycosides

Conjugative transfer

Gram-positive Gram-negative

Soil microorganisms

1965 β-lactamase S. aureus 1965 β-lactamase E. coli

1970 β-lactamase & aminoglycosides S. aureus

Courvalin P, Antimicrob Agent Chemother. 1994;38.

Plasmids

Ü  Integrons are elements containing the genetic determinants of the components of a site-specific recombination system that recognizes and captures mobile gene cassettes.

Ü  Integrase (int) and adjacent recombination sites (attI).

Ü Gene cassette consist of one coding sequence

Integrons & gene cassettes

Although integrons themselves are not mobile, they are sometimes found as part of transposons. These transposons are generally located on plasmids – further enhancing their spread.

Fluit, AC. Eur J Microbiol Infect Dis. 1999;18.

Integrons abound

•  France: 59% in Enterobacteriaceae from clinical specimens (n=49).

•  Germany: 13% in 11 Gram-negative species from blood cultures (n=278).

•  Nine countries: 42% in 13 species of Gram-negative from clinical specimens (n=163).

•  Integrons in staphylococci and enterococci

•  Integrons from primates Fluit, AC. Eur J Microbiol Infect Dis. 1999;18.

Chromosome

Conjugative Plasmid

tra1

oriT

tra2

Transposon

conjugation TetM Van β-lac

Rec

vanR vanW vanY vanB

Integron

Mutation of acquired genes: Structural, β-lactamases

Mutations of acquired genes

Type of mutation Resulting gene / phenotype

Structural ESBL Regulatory Mec (methicillin resistance)

ESBL

Mutation SHV β-lactamase

Group A

TEM & SHV

Pen Cef ’s

Penems

Inhibitor Sensitive

Group C

AmpC

Cef ’s Oxa

Inhibitor Resistant

Group D

OXA

Pen esp Oxa

Inhibitor

S / R

Group B

IMP & VIM

Penems

Inhibitor Resistant

Active site Serine Active site Zn

(metallo)

β-lactamase classification

Evolution of Class A SHV β-lactamases

Ü  Functionality

Ü  Enzyme active-site residues

Ü  3-D conformation

Ü  Expanding substrate range

HSV-2 HSV-5 HSV-8 HSV-9

HSV-4

HSV-12

HSV-1 HSV-10

HSV-11 HSV-2a

HSV-3 HSV-6 ? HSV-7

238

8 35 43 54 130 140 179 192 193 195 205 238 240

SHV-1 Klebsiella 1974 I L R G S A D K L T R G E Pen

SHV-2 Klebsiella, Serratia 1983 S ESBL

SHV-2a Klebsiella 1990 Q S ESBL

SHV-3 Klebsiella 1988 L S ESBL

SHV-4 Klebsiella 1988 L S K ESBL

SHV-5 Klebsiella 1989 S K ESBL

SHV-6 Klebsiella 1991 A CAZ

SHV-7 Escherichia 1995 F S S K ESBL

SHV-8 Escherichia 1997 N ESBL

SHV-9Eschirichia, Klebsiella,

Serratia1995 Del R N V S K ESBL

SHV-10 Eschirichia 1997 Del G R N V S K IR ESBL

SHV-11 Enterobacteriaceae 1997 Q Pen

SHV-12 Enterobacteriaceae 1997 Q S K ESBL

SpectrumAmino acid at positionß-

LactamaseOrigin Country Year

205

240

240

205 179 43

8

179 ?

54, 140

192, 193

130

35 35

238

35

?

?

Heritage, J. J Antimicrob Chemother. 1999;44.

Clonal Non clonal

(gene exchange)

Nosocomial spread of resistance plasmid

Ü Outbreak of ESBL and aminoglycoside-resistant E. cloacae (June – November 2000) followed by…

Ü  Isolation of ESBL and aminoglycoside-resistant A. baumanii (November 2000)

E. cloacae

A. baumanii

Al Naiemi, N. J Clin Microbiol 2005:43

Ü  Infection control measures after E. cloacae outbreak Ü  Hand hygiene Ü  Gloves and gowns during patient care activities Ü  Isolation of patients with MDR E. cloacae infections in

private rooms Ü  These measures failed to prevent the clonal MDR A. baumanii infections

Ü  Other Gram-negative bacteria may have acted as a reservoir of the plasmid

Ü  Infection control measures after A. baumanii infections Ü  Control measures to all patients in ICU Ü  Closure of ICU to new admissions Ü  Dedicated nursing team for patients colonized with the resistant strain

Ü  These measures were successful in halting the MDR A. baumanii outbreak

Ü  Lead to a significant decrease of all MDR-Gram-negative bacilli Al Naiemi, N. J Clin Microbiol 2005:43