1. Mechanisms of Antibiotic Resistance

2. What is antibiotic resistance?
Antibiotic resistance occurs when an antibiotic has lost its ability to effectively control or kill bacterial growth; in other words, the bacteria are "resistant" and continue to multiply in the presence of therapeutic levels of an antibiotic.

3. Why do bacteria become resistant to antibiotics?
When antibiotics are used to kill the bacterial microorganisms, a few microorganisms are able to still survive, because microbes are always mutating, eventually leading to a mutation protecting itself against the antibiotic

4. Antibiotics that are used correctly overwhelm the harmful bacteria
Overuse of antibiotics or unnecessary use creates a selective environment
Resistant bacteria has better fitness in this context
Resistant strains survive and multiply.
After reproducing, the resistant bacteria move to another host.

5. Sex in bacteria Bacteria do exchange genes forming new combinations
Bacteria exchange genes is by conjugation
This involves the transfer of genetic material via a cytoplasmic bridge between the two organisms
This can be done between unrelated species of bacteria
Recent studies on bacteria in the wild show that it definitely occurs in the soil, in freshwater and oceans and inside living organisms

6. The magic bullet Antibiotics revolutionised medicine
The first antibiotic, penicillin, was discovered by Alexander Fleming in 1929
It was later isolated by Florey and Chain
It was not extensively used until the 2nd World War when it was used to treat war wounds
After 2nd World War many more antibiotics were developed
Today about 150 types are used
Most are inhibitors of the protein synthesis, blocking the 70S ribosome, which is characteristic of prokaryotes

7. Resistance
It took less than 20 years for, bacteria to show signs of resistance
Staphylococcus aureus, which causes blood poisoning and pneumonia, started to show resistance in the 1950s
Today there are different strains of S. aureus resistant to every form of antibiotic in use

8. Multiple resistance
It seems that some resistance was already naturally present in bacterial populations
The presence of antibiotics in their environment in higher concentrations increased the pressure by natural selection
Resistant bacteria that survived, rapidly multiplied
They passed their resistant genes on to other bacteria (both disease causing pathogens and non-pathogens)

9. Transposons & Integrons
Resistance genes are often associated with transposons, genes that easily move from one bacterium to another
Many bacteria also possess integrons, pieces of DNA that accumulate new genes
Gradually a strain of a bacterium can build up a whole range of resistance genes
This is multiple resistance
These may then be passed on in a group to other strains or other species

10. Antibiotics promote resistance
If a patient taking a course of antibiotic treatment does not complete it
Or forgets to take the doses regularly,
Then resistant strains get a chance to build up
The antibiotics also kill innocent bystanders bacteria which are non-pathogens
This reduces the competition for the resistant pathogens
The use of antibiotics also promotes antibiotic resistance in non-pathogens too
These non-pathogens may later pass their resistance genes on to pathogens

11. How humans have created the upsurge of bacterial diseases:
International travel
Inadequate sanitation
“antibiotic paradox”

12. Antibiotic Resistance – A Global Problem
VRE
PRP
VISA
MRSA
ESBL
MBL
VRSA
1961
1967
1983
1986
1988
1996
2002
Vancomycin and teicoplanin
Penicillin
Vancomycin
All -lactams
3rd gen cephalosporin
Vancomycin and teicoplanin
Carbapenem
Emergence → Spread

13. How do bacteria become resistant?
Bacteria can gain resistance over time through:
Acquired resistance
Vertical gene transfer
Horizontal gene transfer

14. Genetic Basis of Resistance
Spontaneous mutations in endogenous genes
Structural genes: expanded spectrum of enzymatic activity, target-site modification, transport defect
Regulatory genes: increased expression
Acquisition of exogenous genes
Usually genes that encode inactivating enzymes or modified targets, regulatory genes
Mechanisms of DNA transfer: conjugation (cell–cell contact); transformation (uptake of DNA in solution); transduction (transfer of DNA in bacteriophages)
Expression of resistance genes
Reversible induction/repression systems can affect resistance phenotypes

15. Resistance by Spontaneous Mutations

16. Acquisition of Resistance Genes.

17. Mechanisms of Resistance Gene Transfer

18. Mechanisms of Resistance
Antibiotics exert selective pressure that favours emergence of resistant organisms
Bacteria employ several biochemical strategies to become resistant

19. Major Classes of Antibiotics
Mechanism of action
Major resistance mechanisms
β-Lactams
Inactivate PBPs (peptidoglycan synthesis)
β-lactamases
Low affinity PBPs
Efflux pumps
Glycopeptides
Bind to precursor of peptidoglycan
Modification of precursor
Aminoglycosides
Inhibit protein synthesis (bind to 30S subunit)
Modifying enzymes (add adenyl or Phosphate)
Macrolides
Inhibit protein synthesis (bind to 50S subunit)
Methylation of rRNA
(Fluoro)Quinolones
Inhibit topoisomerases (DNA synthesis)
Altered target enzyme
PBPs penicillin-binding proteins

20. b-Lactams: Classification (1)
Penicillins
Narrow-spectrum penicillins
Broad-spectrum penicillins
β-lactamase inhibitor combinations
Oxacillin derivatives
Cephalosporins (ATC/WHO 2005 classification)
1st generation: Gram-positive cocci (GPCs), some Gram-negative bacilli (GNBs)
2nd generation: some GNBs, anaerobes
3rd generation: many GNBs, GPCs
4th generation: many GNBs resistant to 3rd generation, GPCs

21. b-Lactams: Classification (2)
Carbapenems
Imipenem, meropenem, Doripenem, ertapenem
Monobactams
Aztreonam

22. Mechanism of Action of b-Lactams (1)
Structure of peptidoglycan |
L-Ala
D-Glu
L-diA
D-Ala
NAG-NAM-NAG-NAM
-(AA)n-NH2
Cytoplasm
Transpeptidation reaction

23. Mechanism of Action of b-Lactams (2)
Penicillin-binding proteins (PBPs)
Membrane-bound enzymes
Catalyse final steps of peptidoglycan synthesis (transglycosylation and transpeptidation)
-lactams
Act on PBPs, inhibit transpeptidation
Substrate analogues of D-Ala-D-Ala

24. Resistance to b-Lactams

25. Resistance to b-Lactams
Gram-negative b-lactamases
Major resistance mechanism in nosocomial GNB pathogens
>470 b-lactamases known to date
Classified into 4 groups based on sequence similarity
Ambler Class A (TEM, SHV, CTX), C and D (OXA) are serine b-lactamases
Ambler Class B are metallo-b-lactamases
Their spread has been greatly exacerbated by their integration within mobile genetic elements
Integron-borne b-lactamase genes are part of multi drug resistance gene cassettes
Multidrug-resistant nosocomial pathogens
with complex resistance patterns
Selection of potent b-lactamases
through use of non-b-lactam agents

26. Ambler Classification of β-Lactamases
Active site
Serine-enzymes
Zinc-enzymes
Nucleotide sequence A C D B
Four evolutionarily distinct molecular classes

27. Modified Bush–Jacoby–Medeiros Classification of b–Lactamases

28. b-Lactamases: Classification
Serine enzymes Metallo (Zn) enzymes Group C Group A Group D Group B AmpC TEM/SHV OXA IMP/VIM Cephs Pens, Cephs Pens, esp Oxa Carbapenems Inhib-R Inhib-S Inhib-R/S Inhib-R
Bush. Rev Inf Dis 1987;10:681; Bush et al. Antimicrob Agents Chemother 1995;39:12; Bush. Curr Opin Investig Drugs 2002;3:1284

29. Induction of Group 1 (AmpC) b-Lactamases
Inducible:
Enterobacter spp.
Citrobacter spp.
Morganella spp.
Providencia spp.
Serratia spp.
P. aeruginosa
Amount
enzyme
per cell
Absent :
Salmonella spp.
Klebsiella spp.
Basal :
E. coli
Shigella spp.
b-lactam concentration

30. Selection of Group 1 (AmpC) b-Lactamases
Population of inducible organisms
Derepressed cell due to ampD mutation (Enterobacter : 1 of 105 !)
Ceftazidime, ceftriaxone, piperacillin, etc.:
Selection of derepressed cell
Multiplication and spread of derepressed clone

31. Group 1 (AmpC) b-Lactamases
Produced constitutively in tiny concentrations by certain GNB
Induction of production:
Can occur by the exposure to certain antibiotics (eg, carbapenems)
Only in vitro phenomenon; not clinically relevant (stops when antibiotic use is discontinued; carbapenems not affected by these enzymes)
Selection of production:
Can occur by the use of certain antibiotics (eg, ceftazidime)
Also in vivo phenomenon; highly clinically relevant (does not stop when antibiotic use is discontinued; leads to selection and spread of ABR clones)
Therapeutic options:
4th generation cephalosporins (but resistance may occur with minor AA changes)
Carbapenems

32. Resistance to b-Lactams
Chromosomal AmpC b-lactamases
Several Enterobacteriaceae, including Enterobacter, Citrobacter, and Serratia contain an inducible, chromosomal gene coding for a b-lactamase
Resistant to cephalosporins and monobactams; not inhibited by clavulanate; Class C b-lactamases
Plasmid-mediated AmpC b-lactamases
Arose through transfer of AmpC chromosomal genes into plasmids
Not inducible, with substrate profile (usually) same as parental enzyme
Highly prevalent in the naturally AmpC-deficient K. pneumoniae
Emergence predominantly in community-acquired infections (Salmonella spp., E. coli)
Co-resistance to aminoglycosides, SXT, quinolones
Wide dissemination worldwide (SE Asia, N Africa, South Europe, USA)

33. Plasmid-mediated AmpC b-lactamases (1)
Enzyme Host Country Year isolated
MIR-1 K. pneumoniae US
ACT-1 K. pneumoniae US 1994
E. coli
BIL-1 E. coli UK 1989
CMY-2 K. pneumoniae Greece 1990
S. senftenberg France 1994
Salmonella US 1996
E. coli Libya
Salmonella Spain 1999
Salmonella Romania 2000
LAT-1 K. pneumoniae Greece 1993
LAT-2 K. pneumoniae, Greece 1994
E. coli, E. aerogenes
CMY-3 P. mirabilis France 1998
CMY-4 P. mirabilis Tunisia 1996
E. coli UK 1999
K. pneumoniae Sweden 1998
CMY-5 K. oxytoca Sweden 1988
CMY-7 E. coli India

34. Plasmid-mediated AmpC b-lactamases (2)
Enzyme Host Country Year isolated
DHA-1 Salm. enteritidis Saudi Arabia 1992
K. pneumoniae Taiwan 1999 US
DHA-2 K. pneumoniae France 1992
ACC-1 K. pneumoniae Germany 1997
K. pneumoniae France 1998
P. mirabilis Tunisia 1997
K. pneumoniae Tunisia 1999
Salm. livingstone Tunisia 2000

35. Plasmid-mediated AmpC b-lactamases (3)
Enzyme Host Country Year isolated
FOX-1 K. pneumoniae Argentina 1989
FOX-2 E. coli Germany 1993
FOX-3 K. oxytoca, Italy 1994
K. pneumoniae
FOX-4 E. coli Canaries 1998
FOX-5 K. pneumoniae US
CMY-1 K. pneumoniae Korea 1989
CMY-8 K. pneumoniae Taiwan 1998
CMY-9 E. coli Japan
CMY-10 E. aerogenes Korea
CMY-11 E. coli Korea
MOX-1 K. pneumoniae Japan
MOX-2 K. pneumoniae France

36. Resistance to b-Lactams
Extended-spectrum b-lactamases (ESBL)
No consensus of the precise definition of ESBLs
In general: β-lactamases conferring resistance to the penicillins, 1st , 2nd, 3rd, and even 4th generation cephalosporins, and monobactams, not to carbapenems and cephamycins
Inhibited by b-lactamase inhibitor clavulanic acid
Derived from Class A b-lactamases (exceptions are Class D, OXA): TEM, SHV, CTX-M, OXA, VEB, PER,...
Differ from their progenitors by 1–5 amino acids
Marked and unexplained predilection for Klebsiella pneumoniae
Therapeutic options: carbapenems

37. The Story of E. coli Resistant to Ampicillin
June 1964: ampicillin released in Europe
December 1964; the first case of ampicillin- resistant E. coli detected
Mrs Temoneira (Athens, Greece):
Urinary isolate of E. coli
Produced b-lactamase (TEM-1)
Genes encoding the b-lactamase found on a plasmid

38. The Story of Klebsiella Universally Resistant to Ampicillin
SHV-1 enzyme: b-lactamase with a narrow spectrum of activity (ampicillin)
Chromosomally encoded
If produced in high amounts:
May result in resistance to cefazolin and piperacillin
May even overcome β-lactamase inhibitors (clavulanic acid or tazobactam)

39. The Story of ESBL-producing Enteric GNB
Third generation cephalosporins:
Developed in response to proliferation of K. pneumoniae and E. coli producing b-lactamases active against ampicillin and first generation cephalosporins
Introduced in Europe in the early 1980s
Emergence of extended-spectrum b-lactamases:
Cefotaxime marketed in Germany in September 1981
Cefotaxime-resistant Klebsiella isolate detected in Frankfurt in March 1982 (mutant of the gene encoding SHV-1)

40. Evolution of TEM Enzymes
MIC (mg/mL) ceftazidime
TEM
glutamine arginine
TEM-12
2.0
glutamine serine
TEM-26
128
lysine serine

41. ESBLs in Non-fermenters
Emergence of transferable ESBL enzymes (Class A, B or D) in non-fermenters (P. aeruginosa, Acinetobacter spp.)
ESBL types often different (PER-1, VEB-1, OXA,…) from Enterobacteriaceae
Multiple resistance mechanisms co-expressed (chromosomal AmpC b-lactamase, impermeability, efflux)
Non-fermenters should not be tested routinely for ESBLs
P. aeruginosa: «False-negative» (most ESBLs not inhibited by clavulanate)
Acinetobacter spp.: «False-positive» DD with clavulanate (intrinsic activity of b-lactam inhibitors)
S. maltophilia: «False-positive» DD with clavulanate (inhibition of L2 chromosomal enzyme)

42. FREQUENTLY MADE COMMENT: “I don’t see ESBLs in my hospital”

43. Current ESBL Detection Methods Fail
Routine tests are not designed for ESBL detection
Low level ESBL expression will not be detected by current tests using low inoculum
MIC values and zone sizes of ESBL producers overlap those of susceptible non-ESBL producers
ESBL double disk test may be inaccurate if positioning is suboptimal
ESBL breakpoint methods are limited since MICs for different strains can range over 7 dilutions

44. Resistance to b-Lactams
Carbapenemases
Defined as b-lactamases, hydrolyzing at least imipenem or/and meropenem or/and ertapenem
Belong to Ambler Class A, B, and D, of which Class B are the most clinically significant:
Class A: KPC, SME & NMC/IMI
Class B: IMP, VIM & SPM metallo b-lactamases
Class D: OXA-23, -40 & -58 related

45. Class B (Metallo)-Carbapenemases
Hydrolyzing virtually all b-lactams
Mediate broad spectrum b-lactam resistance
No clinical inhibitor available
Present on large plasmids and integrons
Genes are continuously spreading
Associated (80%) with aminoglycoside resistance
Still rare but increasing, especially in non-fermenters

46. Mobile Carbapenamases
Class I integron
blaIMP
blaVIM
ORF1
aacC4
aacC1
5'cs
3'cs
Nosocomial outbreak of carbapenem-resistant P.aeruginosa and A. baumanii reported in Canada and France, respectively
Cross-resistance to other beta-lactams and to other AB classes
Link with aminoglycoside use, not necessarily carbapenems!

47. Mobile Class B b-Lactamases
Enzyme Host Country (Year)
IMP-1 S. marcescens Japan (>91)
P. aeruginosa Japan
A. xylosoxydans Japan
P. putida Japan
C. freundii Japan
K. pneumoniae Japan,
Singapore (99)
A. baumannii Japan
P. stutzeri, Taiwan
P. putida
A. junii UK (00)
IMP-2 A. baumannii Italy (97)
IMP-3 S. flexneri Japan (96)
IMP-4 Acinetobacter Hong Kong (>94)
C. youngae China (98)
IMP-5 A. baumannii Portugal (98)
IMP-6 S. marcescens Japan (96)
IMP-7 P. aeruginosa Canada (95)
Malaysia (99)
IMP-8 K. pneumoniae Taiwan (98)
IMP-9 P. aeruginosa China (?)
IMP-10 A. xylosoxydans Japan (00)
P. aeruginosa Japan (97)
Enzyme Host Country (Year)
VIM-1 P.aeruginosa Italy (1997)
A. baumannii Italy (1997)
P.aeruginosa Greece (1996)
E. coli Greece (2001)
A. xylosoxydans Italy (1997)
VIM-2 P. aeruginosa France (1996)
P. aeruginosa Greece (1996)
P. aeruginosa Italy (1998)
S. marcescens Korea (2000)
A. baumannii Korea (1998)
P. aeruginosa Belgium (2004/5)
P.putida stutzeri Taiwan (>1997)
VIM-3 P. aeruginosa Taiwan (>1997)
VIM-4 P. aeruginosa Greece (2001)
SPM-1 P. aeruginosa Brazil (1997)
GIM-1 P. aeruginosa Germany (2003)

48. Class D Oxacillinase — Carbapenemases
Class D enzymes
OXA-23, -24, -25, -26, -27, -28, -40, -49, -58, ….
Highly mobile (integron, plasmid)
Found in South America, South-East Asia, Europe (Greece, Spain, Portugal, France, Belgium)
Multi-drug resistance (penicillins and 3rd & 4th generation cephalosporins, BL/BL-inhibitors, aminoglycosides, SXT,…)
Variable resistance levels to imipenem and meropenem (4–>256 mg/mL)

49. Rapidly Increasing Antibiotic Resistance Constitutes One of the Most Important Clinical, Epidemiological and Microbiological Problems of Today