Mechanisms of Resistance

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

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

Major Classes of Antibiotics

104 cfu 4 2 1 0.5 0.25 0.12 0 mg/ml 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

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.

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

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

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

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 ?

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

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.

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

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

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

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?)

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 OM PG porin

Gram-negative TEM-1 b-lactamases • 20-30% of E. coli areampicillin-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 ampicillinresistant • 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).

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

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)

ampCb-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)

ampC Regulation of ampC • 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 + b-lactam-ase

Resistance due to derepression of ampC • Many strains of EnterobacterandCitrobacter 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

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 ampCb-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.

Quinolones • Inhibit topoisomerases/DNA synthesis • 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

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.”

Mechanisms of Resistance