1,Mechanisms of Resistance,X,Efflux,Inactivation,Decreased permeability,Altered target,Antibiotics exert selective pressure that favors emergence of resistant organisms Bacteria employ several biochemical strategies to become resistant,2,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 sequences 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,3,Spread of Resistance Genes,R,S,R,R,S,R,Conjugation,Transformation,R,4,Major Classes of Antibiotics,5,Antibiotic Susceptibility Tests,Minimal inhibitory concentration (MIC) Reference method. Add standard inoculum to dilutions of antibiotic. Incubate overnight. MIC is lowest concentration that inhibits growth (can also be performed by agar dilution). Interpretation (S or R) is based on achievable drug levels,6,Antibiotic Susceptibility Tests,Kirby-Bauer agar disk diffusion Paper disk containing antibiotic is placed on lawn of bacteria, then incubated overnight. Diameter of zone of inhibition is inversely related to MIC (used to establish interpretive breakpoints). Standardized for commonly isolated, rapidly growing organisms.,7,Antibiotic Susceptibility Tests,E-test Strips containing a gradient of antibiotic are placed on lawn of bacteria and incubated overnight. MIC is determined at point where zone of inhibition intersects scale on strip. Combines ease of KB with an MIC method. Particularly useful for S. pneumoniae.,8,b-lactam Antibiotics,Monobactams,Substrate analogs of D-Ala-D-Ala Covalently bind to PBPs, inhibit final step of peptidoglycan synthesis,Penicillins,Carbapenems,Cephalosporins 1st gen: GPC, some GNR 2nd gen: some GNR +anaerobes 3rd gen: many GNR, GPC,9,Structure of Peptidoglycan,cytoplasm,Transpeptidation reaction,10,Penicillin-Binding Proteins (PBPs),Membrane bound enzymes Catalyze final steps of peptidoglycan synthesis (transglycosylation and transpeptidation) Multiple essential PBPs (4-5) - involved in cell elongation, determination of cell shape, and cell division; essential for cell viability -lactams acylate active site serine of PBPs, inhibit transpeptidation Activity determined by affinity for PBPs, stability against -lactamases, and permeability Autolysins contribute to bactericidal activity,11,Penicillin-Resistant S. pneumoniae (PRSP),S. pneumoniae interpretative breakpoints penicillin susceptible (MIC 0.0625 g/ml), intermediate (0.125 -1.0), resistant ( 2.0). High-level penicillin resistance has risen rapidly in US (0.01% in 1987 to 3% in 1994) 20-30% of isolates may be non-susceptible (I or R). High-level PRSP may exhibit cross-resistance to 3rd generation cephalosporins Serious problem when infection occurs at body sites where antibiotic availability is limited. PRSP may be multi-resistant (macrolides, TMP/SXT); strains can spread widely,12,Mosaic PBP Genes in PRSP,Resistance is due to alterations in endogenous PBPs Resistant PBP genes exhibit 20-30% divergence from sensitive isolates (Science 1994;264:388-393) DNA from related streptococci taken up and incorporated into S. pneumoniae genes,Czechoslovakia (1987),USA (1983),South Africa (1978),S SXN,= pen-sensitive S. pneumoniae,= Streptococcus ?,PBP 2B,13,International Spread of PRSP,Multiresistant PRSP in Iceland (JID 1993;168:158-63) First isolate in 12/88; 17% PRSP in 1992. Almost 70% of PRSP were serotype 6B; resistant to tet, chloram, erythro, and TMP/SXT; similar or identical molecular markers. Icelandic PRSP identical to multiresistant 6B clone endemic in Spain (popular vacation site). Possible factors responsible for rapid spread b-lactam use in Iceland low, but high use of TMP/SXT, tet, etc may have selected for multiresistant clone. 57% of population lives in Reykjavik/suburbs, almost 80% of children age 2-6 attend day-care centers.,14,-lactam resistance in Staph. aureus,90% of strains produce -lactamase plasmid encoded, confers resistance to penicillin, ampicillin these strains are susceptible to penicillinase-resistant penicillins (e.g. methicillin), 1st generation cephalosporins, and -lactam/-lactamase inhibitor combinations At many large medical centers, approx 30% of S. aureus are resistant to methicillin and other -lactams,15,Methicillin-resistant S. aureus (MRSA),MRSA contain novel PBP2a, substitutes for native PBPs; low affinity for all -lactams MRSA chromosome contains 50kb mec region not present in MSSA. Acquired from coag-neg Staph spp. PBP2a is encoded by mecA gene; expression controlled by mecI, mecR1 and other f