1. 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

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
Antibiotic Resistance

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

4. 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

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
104 cfu 4 2 1
0.5
0.25
0.12
mg/ml

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 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

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

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
Antibiotic Resistance

11. Penicillin-Resistant S. pneumoniae (PRSP)
S. pneumoniae interpretative breakpoints
penicillin susceptible (MIC  µg/ml), intermediate ( ), 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

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: )
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

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
Antibiotic Resistance

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

16. 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]

17. 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 )

18. 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

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

20. 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

21. 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

22. 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

23. 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

24. -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

25. 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

26. 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

27. 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: )
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

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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: ).
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

34. Quinolone-Resistant Campylobacter jejuni in Minnesota
During resistant isolates increased from 1.3 to 10.2% (NEJM 1999;340: ).
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

35. 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.”