1. 2 antibiotic resistance occurs when an antibiotic has lost its ability to effectively control or...

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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.

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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

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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.

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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

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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

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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

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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)

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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

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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

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International travel

Inadequate sanitation

“antibiotic paradox”

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MRSA MBL

VISA

VRSA

PRP

ESBL

VRE

1961 1967 1983 1986 1988 1996 2002

All -lactams

Penicillin

3rd gen cephalosporin

Carbapenem

Vancomycin Vancomycin

and teicoplanin Vancomycin

and teicoplanin

Emergence → Spread

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Bacteria can gain resistance over time through:

•Acquired resistance

•Vertical gene transfer

•Horizontal gene transfer

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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

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Antibiotics exert selective pressure that favours emergence of resistant organisms

Bacteria employ several biochemical strategies to become resistant

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AntibioticAntibiotic Mechanism of actionMechanism of actionMajor resistance mechanismsMajor resistance mechanisms

ββ-Lactams-Lactams Inactivate PBPs Inactivate PBPs (peptidoglycan synthesis)(peptidoglycan synthesis)

• ββ-lactamases-lactamases• Low affinity PBPsLow affinity PBPs• Efflux pumpsEfflux pumps

GlycopeptidesGlycopeptides Bind to precursor of Bind to precursor of peptidoglycanpeptidoglycan

• Modification of precursorModification of precursor

AminoglycosidesAminoglycosides Inhibit protein synthesis Inhibit protein synthesis (bind to 30S subunit)(bind to 30S subunit)

• Modifying enzymes (add Modifying enzymes (add adenyl or Phosphate)adenyl or Phosphate)

MacrolidesMacrolides Inhibit protein synthesis Inhibit protein synthesis (bind to 50S subunit)(bind to 50S subunit)

• Methylation of rRNAMethylation of rRNA• Efflux pumpsEfflux pumps

(Fluoro)Quinolones(Fluoro)Quinolones Inhibit topoisomerases Inhibit topoisomerases (DNA synthesis)(DNA synthesis)

• Altered target enzymeAltered target enzyme• Efflux pumpsEfflux pumps

PBPs penicillin-binding proteins

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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

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Carbapenems Imipenem, meropenem, Doripenem,

ertapenemMonobactams

Aztreonam

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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

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Gram-negative -lactamases Major resistance mechanism in nosocomial GNB

pathogens >470 -lactamases known to date Classified into 4 groups based on sequence similarity

▪ Ambler Class A (TEM, SHV, CTX), C and D (OXA) are serine -lactamases

▪ Ambler Class B are metallo--lactamases Their spread has been greatly exacerbated by their

integration within mobile genetic elements Integron-borne -lactamase genes are part of multi drug

resistance gene cassettes

Multidrug-resistant nosocomial pathogens with complex resistance patterns

Selection of potent -lactamases through use of non--lactam agents

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Active site

Nucleotide sequence

Four evolutionarily distinct molecular classes

A C D

Serine-enzymes

B

Zinc-enzymes

β-lactamases

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Functional Substrate profile Group

Molecular Class

Inhibitor

Example

1 Cephalosporinase C Oxa AmpC, MIR-1

2a Penicillinase A Clav. S.aureus

2b Broad spectrum A Clav. TEM-1/2, SHV-1

2be Extended spectrum A Clav. TEM 3-29, TEM46-104 SHV2-28, CTX-M types

2br Inhibition resistant A - TEM 30-41 (IRT1-12)

2c Carbenicillinase A Clav. PSE-1

2d Oxacillinase D (Clav.) OXA-1 (OXA-2 &-10 derived ESBL)

2e Cephalosporinase A Clav. FPM-1 P. vulgaris, CepA B. fragilis.

2f Carbapenemase A Clav. IMI-1, NmcA, Sme 1-3

3 Metallo-enzyme B - S.maltophilia

4 Penicillinase - - B.cepacia

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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 CarbapenemsInhib-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

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Inducible: Enterobacter spp.Citrobacter spp.Morganella spp.Providencia spp.Serratia spp.P. aeruginosa

Basal : E. coliShigella spp.

-lactam concentration

Amountenzymeper cell

Absent : Salmonella spp.Klebsiella spp.

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Population of inducible organisms

Derepressed cell due to ampD mutation (Enterobacter : 1 of 105 !)

Selection of derepressed cell

Multiplication and spread of derepressed clone

Ceftazidime, ceftriaxone, piperacillin, etc.:

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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

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Chromosomal AmpC -lactamases Several Enterobacteriaceae, including Enterobacter,

Citrobacter, and Serratia contain an inducible, chromosomal gene coding for a -lactamase

Resistant to cephalosporins and monobactams; not inhibited by clavulanate; Class C -lactamases

Plasmid-mediated AmpC -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)

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Enzyme Host Country Year isolatedMIR-1 K. pneumoniae US 1988ACT-1 K. pneumoniae US 1994

E. coliBIL-1 E. coli UK 1989CMY-2 K. pneumoniae Greece 1990

S. senftenberg France 1994Salmonella US 1996E. coli Libya 1996Salmonella Spain 1999Salmonella Romania 2000

LAT-1 K. pneumoniae Greece 1993LAT-2 K. pneumoniae, Greece 1994

E. coli, E. aerogenesCMY-3 P. mirabilis France 1998CMY-4 P. mirabilis Tunisia 1996

E. coli UK 1999K. pneumoniae Sweden 1998

CMY-5 K. oxytoca Sweden 1988CMY-7 E. coli India

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Enzyme Host Country Year isolatedDHA-1 Salm. enteritidis Saudi Arabia 1992

K. pneumoniae Taiwan 1999 US 1996-2000

DHA-2 K. pneumoniae France 1992ACC-1 K. pneumoniae Germany 1997

K. pneumoniae France 1998P. mirabilis Tunisia 1997K. pneumoniae Tunisia 1999Salm. livingstone Tunisia 2000

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Enzyme Host Country Year isolatedFOX-1 K. pneumoniae Argentina 1989FOX-2 E. coli Germany 1993FOX-3 K. oxytoca, Italy 1994

K. pneumoniaeFOX-4 E. coli Canaries 1998FOX-5 K. pneumoniae US 1999CMY-1 K. pneumoniae Korea 1989CMY-8 K. pneumoniae Taiwan 1998CMY-9 E. coli Japan 1995CMY-10 E. aerogenes Korea 1999CMY-11 E. coli Korea 1998MOX-1 K. pneumoniae Japan 1991MOX-2 K. pneumoniae France 1999

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Extended-spectrum -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 -lactamase inhibitor clavulanic acid Derived from Class A -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

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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 -lactamase (TEM-1) Genes encoding the -lactamase found

on a plasmid

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SHV-1 enzyme: -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)

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Third generation cephalosporins: Developed in response to proliferation of K.

pneumoniae and E. coli producing -lactamases active against ampicillin and first generation cephalosporins

Introduced in Europe in the early 1980s Emergence of extended-spectrum -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)

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MIC (g/mL) ceftazidime

102 162TEM-1 0.25

glutamine arginine

TEM-122.0

glutamine serineTEM-26

128lysine serine

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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 -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 -lactam inhibitors) S. maltophilia: «False-positive» DD with clavulanate (inhibition

of L2 chromosomal enzyme)

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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

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Carbapenemases Defined as -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 -lactamases ▪ Class D: OXA-23, -40 & -58 related

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Hydrolyzing virtually all -lactamsMediate broad spectrum -lactam

resistanceNo clinical inhibitor availablePresent on large plasmids and

integronsGenes are continuously spreadingAssociated (80%) with

aminoglycoside resistance Still rare but increasing, especially in non-fermenters

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ORF1 aacC4 aacC1

blaIMP

blaVIM

Class I integron

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!

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Enzyme Host Country (Year)IMP-1 S. marcescens Japan (>91)

P. aeruginosa Japan A. xylosoxydans JapanP. putida JapanC. freundii JapanK. pneumoniae Japan,

Singapore (99)A. baumannii JapanP. stutzeri, TaiwanP. putida A. junii UK (00)

IMP-2 A. baumannii ItalyItaly (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 FranceFrance (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)

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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 g/mL)

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Rapidly Rapidly IIncreasing ncreasing AAntibiotic ntibiotic RResistance esistance CConstitutes onstitutes OOne of the ne of the

MMost ost IImportant mportant CClinical, linical, EEpidemiological and pidemiological and MMicrobiological icrobiological

PProblems of roblems of TTodayoday

1. 2 antibiotic resistance occurs when an antibiotic has lost its ability to effectively control or...

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