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ANTIBIOTIC RESISTANCE
LauraLe Dyner MD
Pediatric Infectious Disease Fellow
June 2009
Antibiotic Resistance
Relative or complete lack of effect of an antimicrobial agent n An organism is able to grow in readily achievable serum concentrations of the antibiotic in question n n Increase in MIC/Decrease in disk diffusion
Minimum Inhibitory Concentration
(MIC)
Lowest concentration of antimicrobial that inhibits the growth of the organism after an 18 to 24 hour incubation period n Interpreted in relation to the specific antibiotic and achievable drug levels n Can not compare MICs between different antibiotics n Discrepancies between in vitro and in vivo
n MIC/Disk Diffusion
Antibiotic Resistance
n Intrinsic resistance Occurs naturally in all or most strains of that species q Chromosomally encoded q Gram­negatives are resistant to Vancomycin q n Acquired resistance q Results from a mutation in the existing DNA of an organism or acquisition of new DNA
Timeline of Antibiotic Resistance
Factors That Promote Resistance
Exposure to sub­optimal levels of antimicrobials n Exposure to broad­spectrum antibiotics n Exposure to microbes carrying resistant genes n Lack of hygiene in clinical environments n Use of antibiotics in foods/agriculture
n Inappropriate Antimicrobial Use
Prescriptions not taken for a total duration of therapy n Antibiotics for viral infections n Antibiotics sold without medical supervision
n CDC Estimates
Consequences of Increased Resistance
Infections that are difficult to treat n Increased mortality n Increased cost of therapy n MDR­TB q Cost per tablet q n n n PCN $0.24 Linezolid $86.90 360 x as much
Genetic Basis of Resistance
n Chromosomal n n n Mutation Chromosomally mediated inducible enzymes Plasmid n n n n Most common genetic basis of resistance Genetic determinants can spread laterally through a population without cell division Interspecies lateral transfer of plasmids Resistance usually involves antibiotic inactivating enzymes (many encoded by transposons)
Selection of Resistant Bacteria
Mechanisms of Resistance
Enzymatic Inactivation** n Decreased permeability n Efflux n Alteration of target site n Overproduction of target
n Decreased Permeability
n Outer membrane permeability Gram­negative bacteria q Impedes the entry of hydrophobic antibiotics (nafcillin/erythromycin) q Porins q q n Loss of porins leads to increased resistance to b­lactams Inner membrane permeability q Altered proton motive force leading to aminoglycoside­resistance after long term use
Efflux
n Tetracyclines q Energy­dependent transporter system Fluoroquinolones n Chloramphenicol n Macrolides n mef gene (macrolide efflux) q S pneumo, S aureus, S epidermidis
q Target Sites
1955 1962 1985 1940
1990 2000 1959 1950 1962 1955 1948 1944 1947 1963 Altered target sites
n Cell wall PBPs: S pneumo, N meningitidis, MRSA q D­Ala­D­Ala target: VRE q n VanA, VanB, VanC, VanD Cell membrane changes n Ribosomal alterations n Tetracyclines, macrolides, lincosamides, aminoglycosides q Failure of the antibiotic to bind to the ribosome disrupts the ability to inhibit protein synthesis
q Penicillin Binding Proteins (PBP)
n More common resistance mechanism for gram­positive organisms n Gram­negative access to PBPs is limited due to the outer membrane Target for all b­lactams n Found as membrane­bound & cytoplasmic proteins n Involved in the final stages of peptidoglycan synthesis of cell walls
n Inactivating enzymes
b­lactamases** n Aminoglycoside modifying enzymes n Chloramphenicol acetyltransferase n Macrolide, Lincosamide, & Streptogramin inactivating enzymes n Tetracycline inactivation n
tetX enzyme q Although most tetracycline resistance is mediated by efflux & ribosomal protection
q b-lactamases
Large, diverse family of enzymes n Wide range of activity n ESBLs q AmpC b­lactamases q Carbapenemases q n Widely dispersed Gram­positive q Gram­negative q n q Major mechanism of resistance in gram­negatives Anaerobes
b-lactam Mechanism of Action
b­lactam antibiotic binds to PBP n Inhibition of peptidoglycan synthesis n
q n Inhibits synthesis of cross­links Cell death
b-lactamases
q Split the amide bond of the b­lactam ring
b-lactamases: Structural Classes
n n n n Class A n Plasmid mediated n Serine residue at the active site n Preferentially hydrolyze penicillins Class B n Chromosomal n Metalloenzymes; zinc­binding thiol group n Hydrolyze carbapenems, penicillins, & cephalosporins Class C n Chromosomal n AmpC n Mainly active against cephalosporins Class D n Plasmid mediated n Oxacillin­hydrolyzing enzymes
b-lactamases: Classification
AmpC
n Confers resistance to Cephamycins (cefotetan, cefoxitin) q Oxyimino­b­lactams (ceftriaxone, cefotaxime, ceftazidime) q Chromosomal in SPACE organisms & are inducible n Plasmid mediated in other gram­negatives & usually not inducible n Not inhibited by b­lactamase inhibitors
n AmpC
Carbapenemases
n n Can hydrolyze penicillins, cephalosporins, monobactams, & carbapenems. Groups q Metallo­ b­lactamases (MBLs) n n q Confer a high level of resistance Pseudomonas, Acinetobacter, Enterobacter Serine­ b­lactamases n Oxacillinases or D b­lactamases (OxaA) q q q n Not as diverse Acinetobacter Another resistance is usually necessary to raise the MIC Class A Carbapenemases q q Pseudomonas & Enterobacter Klebsiella: predominant type (plasmid)
ESBL Mediated Resistance
n Contain a number of mutations that allow them to hydrolyze expanded­spectrum β­lactam antibiotics n Derived from older antibiotic­hydrolyzing b­ lactamase enzymes (TEM­1, TEM­2, SHV­1) q q q q n A single amino acid substitution can give rise to new ESBLs Not as catalytically efficient Inhibited by β­lactamase inhibitors Susceptible to cefoxitin and cefotetan in vitro only 10%–40% of K pneumoniae, E coli express ESBLs
Distinction between AmpC & ESBL
n The AmpC b ­Lactamases are encoded by genes located on chromosomes q q q n n n Often inducible Commonly found in Enterobacter sp, Citrobacter freundii, Morganella morganii, Serratia marcescens, and Pseudomonas aeruginosa. The genes are not easily transferable to other bacterial species ESBLs are encoded by genes located on plasmids, resulting in easy transfer to other bacterial species. AmpC b ­Lactamases are weakly inhibited by b ­ Lactamase inhibitors (clavulanic acid) and usually confer resistance to cephamycins In contrast, ESBLs are generally well inhibited by b ­ Lactamase inhibitors and usually retain sensitivity to the cephamycins (in vitro)
Resistance Against Specific Antibiotics
Beta­lactams β ­lactamases Alteration of PBPs Aminoglycosides Enzyme modification of the drug ­ common Ineffective transport Altered ribosomal binding site (30S) – rare Macrolides Decreased permeability of cell wall to drug Alteration in the 50S ribosomal binding site Inactivation by enzymatic hydrolysis (esterase) Efflux pump Sulfonamides Overproduction of PABA Trimethoprim Plasmid mediated dehydrofolate reductases Vancomycin Low frequency of resistance
Resistance Against Specific Antibiotics
Cephalosporins Extended Spectrum Beta Lactamases (ESBL) Chromosomal cephalosporinases
b­Lactamase Inhibitors Hyperproducers of b­lactamases New b­lactamases resistant to inhibitors Chromosomal cephalosporinases Carbapenems Porin mutations Efflux pump overproduction (excluding Imipenem) Zinc Metalloenzymes and other b­lactamases Fluoroquinolones Decreased number of porins in target cells Efflux mechanisms Mutations in DNA gyrase & topoisomerase)
Specific Organisms
n Staphylococcus aureus n n n MRSA VISA VRSA Group A (beta­hemolytic) Strep n Streptococcus pneumoniae n Enterococcus n n n VRE “SPACE” (Serratia, Pseudomonas, Proteus, Acinetobacter, Citrobacter, Enterobacter)
Staph Aureus
n Produce b­lactamases Preferentially hydrolyze penicillins q Encoded for on bacterial plasmids or transposons q At LPCH approximately 18% of S aureus is susceptible to penicillin or ampicillin n Methicillin & nafcillin are resistant to penicillinase n Resistance has increased in recent years
n Methicillin Resistant Staph Aureus
(MRSA)
1959: n 1961: n 1968: n 1974: n 1981: n 2002: n n 2007: Methicillin introduced MRSA discovered in England MRSA found in the US (Boston) 2% of hospital staph infections MRSA acquired in the community Community & hospital acquired infections are found to be genetically different In the US: 94,000 severe infections/yr & 19,000 deaths/yr
MRSA
Most frequent nosocomial resistant pathogen n Acquired via MecA n Chromosomal mutation leading to the synthesis of a new penicillin binding protein n The protein is inducible & confers resistance to all beta lactam antibiotics
n MecA
n Encodes for PBP 2a q n n Weak affinity for methicillin & all β­lactams Speculation that the origin was from coagulase­ negative Staph or E coli Mobile chromosomal element called staphylococcal cassette chromosome (SCCmec) n SCCmec types I, II, and III q q q n Multidrug resistant Large cassettes Health­care associated SCCmec type IV and type V q q Not multidrug resistant Community associated
Clindamycin Resistance: D-test
n Resistance to macrolides (e.g. erythromycin) can occur by two different mechanisms Mechanism Clinda Efflux Gene msrA Ribosome Alt. erm erm Erythro R S R R S* R
Clindamycin Resistance: D-test
VISA/VRSA
n VISA: Vancomycin Intermediate Staph Aureus q n VRSA: Vancomycin Resistant Staph Aureus q n MIC 4­8 ug/ml MIC > 16 ug/ml Glycopeptide resistance
Group A Strep
100% susceptible to penicillin & other beta lactam antibiotics n Some are resistant to macrolides n q In a European surveillance, it has been found to be as high as 1/3 resistant
Streptococcus pneumoniae
n Resistance to penicillin due to stepwise mutations in penicillin binding proteins (PBP) q q q q n n Chromosomally mediated Decreased affinity to PBP Can be overcome with high­dose penicillin Alterations in PBPs decrease susceptibility of the organism to other beta lactam antibiotics Macrolides q Genetic changes to binding target on ribosome­high level can not be overcome = erm(B) q Efflux pump­lower level­may be overcome = mef (A) Vancomycin is used to treat penicillin & cephalosporin resistant infections
Enterococcus
n Intrinsic resistance q q q q n Chromosomal Less susceptible to penicillin & ampicillin than other streptococci (MIC 2­8 ug/ml) [by 10­1000 x] Low level of resistance to aminoglycosides Synergy between a cell wall agent & an aminoglycoside Acquired resistance q q Beta lactamase production High level of resistance to aminoglycosides n q Lose synergy ability as well Glycopeptide resistance (Vancomycin)
Vancomycin Resistant Enterococci
(VRE)
n n First reported in 1988 Origins q q q q n n Normal fecal flora Food chain Selection by use of Vancomycin, Cephalosporins, Anaerobic Antibiotics Person­to­person spread Produce a new membrane protein to inhibit binding of vancomycin to D­alanyl­D alanine terminus Lack of synergy with aminoglycosides
Vancomycin Resistant Enterococci
(VRE)
“SPACE” Organisms
Gram­negative bacteria have a chromosomal gene for the b ­lactamase, ampC n SPACE organisms can undergo single­step mutations to constitutive, high­level enzyme production n If enough ampC beta­lactamase is produced, resistance occurs
n Conclusion
n Combat Antibiotic Resistance! Narrow antibiotic coverage when possible n Restrict antimicrobial use n q At LPCH: Linezolid, Micafungin, Caspofungin Track resistance patterns n Direct Observed Therapy (TB)
n Resources
n n n n n n n Centers for Disease Control Johns Hopkins Antibiotic Guide UpToDate 2009 Nature The 2006 American Academy of Pediatrics Redbook Mandell’s Prober, Long, & Pickering. Principles & Practice of Pediatric Infectious Disease, 3 rd Edition