Mechanisms of Resistance Antibiotics exert selective pressure that favors emergence of resistant organisms Bacteria employ several biochemical strategies to become resistant Decreased permeability Inactivation Efflux X Altered target

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 Antibiotic Resistance

Spread of Resistance Genes Conjugation R S R R Transformation S R R

Major Classes of Antibiotics Mechanism of action Major resistance mechanisms Beta-lactams Inactivate PBPs (peptidoglycan synthesis) Beta-lactamases Low affinity PBPs Decreased transport Glycopeptides Bind to precursor of peptidoglycan Modification of precursor Aminoglycosides Inhibit protein synthesis (bind to 30S subunit) Modifying enzymes (add adenyl or PO4) Macrolides Inhibit protein synthesis (bind to 50S subunit) Methylation of rRNA Efflux pumps Quinolones Inhibit topoisomerases (DNA synthesis) Altered target enzyme

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 104 cfu 4 2 1 0.5 0.25 0.12 mg/ml

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.

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.

b-lactam Antibiotics Substrate analogs of D-Ala-D-Ala Covalently bind to PBPs, inhibit final step of peptidoglycan synthesis N O S N O S Cephalosporins 1st gen: GPC, some GNR 2nd gen: some GNR +anaerobes 3rd gen: many GNR, GPC Penicillins N O N O Carbapenems Monobactams Antibiotic Resistance

Structure of Peptidoglycan cytoplasm | L-Ala D-Glu L-diA D-Ala NAG-NAM-NAG-NAM -(AA)n-NH2 Transpeptidation reaction Antibiotic Resistance

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 Antibiotic Resistance

Penicillin-Resistant S. pneumoniae (PRSP) S. pneumoniae interpretative breakpoints penicillin susceptible (MIC  0.0625 µg/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 Antibiotic Resistance 10

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 S SXN PBP 2B Czechoslovakia (1987) South Africa (1978) USA (1983) = pen-sensitive S. pneumoniae = Streptococcus ? Antibiotic Resistance

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.

-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 Antibiotic Resistance

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 factors. Most MRSA are also resistant to macrolides and fluoroquinolones; remain susceptible to vancomycin. Major nosocomial pathogen; primarily spread on hands of healthcare workers.

Enterococci and -lactams Intrinsically less susceptible to -lactams PenG/Amp MICs 10-fold higher than other streptococci, not bactericidal PenG/Amp + gent (bactericidal) for endocarditis Ampicillin alone effective for UTI Cephalosporins not active; increase risk of enterococcal infection Acquired resistance is a new problem High-level Amp resistance (altered PBPs in E. faecium); [-lactamase still rare]

Vancomycin-resistant Enterococci Since 1989, a rapid increase in the incidence of infection and colonization with vancomycin-resistant enterococci (VRE) has been reported by U.S. hospitals (MMWR Vol. 44 / No. RR-12) This poses important problems, including: Lack of available antimicrobial therapy for VRE infections because most VRE are also resistant to drugs previously used to treat such infections (e.g. aminoglycosides and ampicillin). Possibility that vancomycin-resistance genes present in VRE can be transferred to other gram-positive bacteria (e.g. Staph. aureus )

Vancomycin Member of glycopeptide family Binds to D-Ala-D-Ala in peptidoglycan precursors Prevents transglycosylation and transpeptidation Resistance to -lactams does not confer cross-resistance to vancomycin Only active against Gram-positives Cannot cross outer membrane of Gram-negatives Primarily used for MRSA, MRSE infections; pts with penicillin allergy; severe C. difficile disease. Antibiotic Resistance 23

Mechanism of Action of Vancomycin G M G M G M G Vancomycin binds to D-Ala-D-Ala; prevents transglycosylation and transpeptidation Vancomycin Antibiotic Resistance 24

Mechanism of VRE Acquired high-level resistance E. faecium and E. faecalis containing vanA or vanB gene clusters produce modified peptidoglycan containing D-Ala-D-lactate; does not bind vancomycin (MIC = 32 - >256 Resistance genes are on mobile elements, have spread widely since 1st reports in late 80’s; major focus of infection control Multiresistant E. faecium (vancomycin, high-level ampicillin, high-level aminoglycoside) poses therapeutic challenge Other enterococci contain vanC; low-level, non-transferable resistance; strains have low pathogenicity Antibiotic Resistance

Vancomycin Resistance pyruvate M G M G vanH vanA + D-Lac D-Ala ddl vanX + Vancomycin does not bind to modified peptidoglycan Antibiotic Resistance 28

Epidemiology of VRE Risk factors for colonization/infection in USA Severe underlying disease (malignancy, ICU, long hosp); antibiotics (vancomycin, 3rd gen cephs) Reservoirs, routes of dissemination not fully understood VRE strains can be distinguished by molecular typing (PFGE) Multiple patterns are seen in some institutions (endogenous infection from intestinal source?) Clonal outbreaks are seen in others (transmission by HCWs?, fomites?) Antibiotic Resistance

VRSA - An emerging Problem Several reports of S. aureus with reduced susceptibility to vancomycin since 1997 Japan and U.S. (Michigan, NJ, NY, Illinois) Vancomycin MIC = 8 g/ml Isolates obtained from patients with chronic MRSA infection No evidence of vanA or vanB Decreased susceptibility due to increased levels of peptidoglycan and precursors

-lactam resistance in Gram-negative rods Factors that increase the MIC (resistance) Increased enzymatic inactivation High VMAX and/or low KM Increased enzyme concentration Decreased intracellular concentration Decreased influx Increased efflux) Multiple mechanisms may function in the same strain PM PG OM porin

Gram-negative TEM-1 b-lactamases 20-30% of E. coli are ampicillin-resistant Most contain a plasmid-encoded class 2b b-lactamase (TEM-1). Active against penicillins but not 3rd generation cephalosporins. Inhibited by clavulanate. All K. pneumoniae are ampicillin resistant Contain chromosomal SHV-1 (related to TEM-1) Most E. coli and K. pneumoniae are susceptible to 1st gen cephs (e.g. cefazolin). Until recently, all were susceptible to 3rd gen cephalosporins (e.g. ceftriaxone, ceftazadime). Antibiotic Resistance

Extended Spectrum Beta-lactamases (ESBLs) Changes in 1-5 amino acids near active site serine of TEM-1 (or SHV-1) greatly increase activity against 3rd gen cephalosporins and monobactams. TEMs 3-29, SHVs 2-6; still inhibited by clavulanate Carbapanems are only reliable b-lactams vs ESBL producers Mainly seen in E. coli and K. pneumoniae Located on transferable plasmids that may carry additional resistance genes Antibiotic Resistance

Ceftazidime, Imipenem and ESBLs During early 1990s, ESBL-producing Klebsiella became increasingly common at a hospital in NYC. In 1996 cephalosporin use was sharply curtailed to attempt reduce the ESBL burden (JAMA 1998;280:1233-37) 1995 1996 Median monthly use (grams) Ceftazidime 383 66 Imipenem 197 474 Ceftazidime-resistant Klebsiella (nososcomial) 150 84 Imipenem-resistant P. aeruginosa (nosocomial) 67 113

ampC b-lactamases Several Enterobacteriaceae, including Enterobacter, Citrobacter , and Serratia, contain an inducible, chromosomal gene coding for a b-lactamase (ampC) Very active in vitro against 1st gen cephs; low activity against 3rd gen cephs; not inhibited by clavulanate These organisms are naturally resistant to cefazolin, cefoxitin (strong inducers of ampC) Usually sensitive to 3rd gen cephs (poor inducers of ampC) Antibiotic Resistance

Regulation of ampC + + b-lactam-ase Recycling of peptidoglycan produces NAM-tripeptide Normally catabolized by AmpD (NAM-tripeptide amidase) and recycled into new peptidoglycan Peptidoglycan Peptidoglycan autolysins AmpD + [AmpR]- [AmpR]+ NAM-tripeptide is also a positive activator of AmpR Increases transcription of ampC + ampC b-lactam-ase Antibiotic Resistance

Resistance due to derepression of ampC Many strains of Enterobacter and Citrobacter develop resistance to 3rd gen cephs during therapy. Resistant variants contain mutations that inactivate AmpD NAM-tripeptide accumulates, causes stable derepression of ampC Increased levels of AmpC b-lactamase inactivates 3rd gen cephalosporins Resistant strains remain susceptible to imipenem (a carbapenem) Poorly hydrolyzed, targets low copy PBP Antibiotic Resistance

b-lactam Resistance in P. aeruginosa Naturally resistant to many antibiotics Outer membrane lacks high permeability porins present in Enterobacteriaceae. Pump mechanism actively exports antibiotics Acquired resistance is common Inducible ampC b-lactamase Imipenem resistance due to mutations that inactivate porin D2 (basic AA transporter) Sole transporter of imipenem Mutations in D2 decrease imipenem influx; b-lactamase inactivates sufficient drug to confer resistance. Antibiotic Resistance

Quinolones Inhibit topoisomerases/DNA synthesis Acquired resistance Trap enzyme-DNA complex after strand breakage DNA gyrase (topo II) (gyrA/gyrB) Primary target in Gram-negatives Topoisomerase IV [parC/parE (grlA/grlB in S.aur)] Primary target in Gram-positives Acquired resistance Mutations in DNA gyrase and topo IV subunits Mainly gyrA and parC (grlA) Stepwise increase in resistance results from sequential mutations in primary and secondary targets Efflux pumps P. aeruginosa, S. aureus, S. pneumoniae

Rapid Appearance of Ciprofloxacin Resistance in S. aureus After the introduction of ciprofloxacin in the late ’80s there was rapid increase in resistance among MRSA Prior to introduction of cipro at the Atlanta VAMC, 0% of MRSA were cipro-resistant. One year after introduction, 79% of MRSA were cipro-resistant (JID 1991;163:1279-85). More than one clone developed resistance One-half of pts had been previously treated with cipro (given for other infections) One year later 91% were resistant 13% of MSSA also became cipro-resistant

Quinolone-Resistant Campylobacter jejuni in Minnesota During 1992-8 resistant isolates increased from 1.3 to 10.2% (NEJM 1999;340:1525-32). Foreign travel was the major risk factor Mexico, Caribbean, Asia Prior antibiotic therapy accounted for 15% of resistance There was also an increase in domestically acquired resistant isolates that was temporally related to the introduction of quinolones for treatment of poultry in 1995 Quinolone-resistant isolates were cultured from 20% (18/91) of retail chicken products 6/7 resistant subtypes (PCR-RFLP) from chicken were also isolated from humans Antibiotic Resistance

Optimism and Concern on Many Fronts NEJM (8/14/97): “In Finland, after nationwide reductions in the use of macrolide antibiotics for outpatient therapy, there was a significant decline in the frequency of erythromycin resistance among group A streptococci isolated from throat swabs and pus samples.” NEJM (9/4/97): “We report high-level resistance to multiple antibiotics, including all the drugs recommended for plague prophylaxis and therapy, in a clinical isolate of Y. pestis. The resistance genes were carried by a plasmid that could conjugate to other Y. pestis isolates.”