1. Interpretation of the antibiogram : from the lab to the bedside
Maria Virginia Villegas , MD, MSc,FIDSA
Scientific Director Bacterial Resistance and Hospital Epidemiology Research Area
International Center for Medical Research and Training, Cali, Colombia CIDEIM

2. MECHANISMS OF RESISTANCE IN GRAM (-) BACTERIA
Peleg AY, Hooper DC. Hospital-acquired infections due to gram-negative bacteria. N Engl J Med. 2010 May 13

3. MECHANISMS OF RESISTANCE IN GRAM (-) BACTERIA
Cephalosporins
slower diffusion due to bulk
and ionic charges
Imipenem
Rapid diffusion due to small size
and zwitterionic (+/-) charge
Outer Membrane
(Lipopolysaccharide
mutated or missing D2 porin
OmpF
OmpC
Periplasmic Space
efflux
pump
This represents a graphic of the periplasmic space and the target sites for antimicrobial activity in gram-negative organisms.
Also identified are the penetration methods for the various antibiotics depicted.
Special thanks to Rich Reinert for this slide.
beta lactamases
(hydrolyzing enzymes)
penicillin binding proteins
Inner Membrane
(Phosolipid)
PBP1b
PBP3
PBP2
PB1a

4. Mechanisms of Resistance in Gram (-) Bacteria
Altered permeability to antibiotics
Changes in the Outer Membrane and Porins
Efflux
Target changes ( PBP, Ribosome, DNA )
Beta-lactamase production
Torres JA, Villegas MV, Quinn JP. Current concepts in antibiotic-resistant gram negative bacteria. Expert Rev Anti Infect Ther 2007; 5(5):
- Briceño DF, Ruiz SJ, Villegas MV. Resistencia a antimicrobianos, Enfermedades Infecciosas CIB, 7º Edición. 2011

5. P.aeruginosa Amikacin S Gentamicina R Cefoperazone/S R Ceftazidime S
Cefepime S
Imipenem R
Ciprofloxacina S
Meropenem S
Doripenem S
Pip/tazobactam S

6. OprD and P.aeruginosa

7. Different Porins and its role in Resistance
Figure: Livermore/ pg 1
OprD selective for carbapenems
Cefalosphorins
Porin
Outer membrane
This diagram depicts the entry of various carbapenems into a bacterium. Through the OprD (initially called D2 porin) and its subsequent interaction with penicillin-binding proteins (PBPs) on the surface of the inner cytoplasmic membrane, P aeruginosa resistance typically invovles loss of OprD (seen here) and over expression of the efflux pump system (not depicted).
Reference:
1. Livermore MD. The microbiology of ertapenem, a new carbapenem. Infections in Oncology. 2001;5(3):1–5.
PBP4
PBP7
PBP1
PBP2
PBP3
Cytoplasmic membrane
Penicillin-binding proteins (PBPs)
Livermore DM. Infections in Oncology. 2001;5:1–5.

8. P.aeruginosa Amikacin S Gentamicina R Cefoperazone/S R Ceftazidime S
Cefepime S
Imipenem S
Ciprofloxacina S
Meropenem R
Doripenem S
Pip/tazobactam S

9. Efflux Transporter (AcrB)
Efflux pumps
Figure:
Nikaido pg 216 OM
Channel (TolC)
Porin
Outer Leaflet (LPS)
Outer Membrane
Inner Leaflet
Periplasmic
Accessory
Protein (AcrA)
Periplasm
Efflux pumps play an important role in the multidrug resistance of P aeruginosa and Acinetobacter. Efflux pumps can remove a wide range of antibiotic drugs from Gram-negative bacteria [Aeschlimann/ pg 916/ A].1 All efflux pump systems are energy-dependent (ie, proton motive force-driven) and they consist of an exporter protein located in the cytoplasmic membrane, a gated outer membrane protein (also called porin) located in the outer membrane, and a membrane fusion protein (linker protein) that links the exporter protein with the outer membrane channel protein [Aeschlimann/ pg 917/ A]. 1 Antibiotics can be captured from the periplasm, the cytoplasmic membrane, and/or the cytoplasmic space by the exporter protein [Aeschlimann/ pg 917/ C]. 1 The accessory proteins serve as a conduit between the cytoplasmic membrane and the outer membrane. Gated outer membrane proteins (porin) serve as the final step in the removal of the antibiotic from the cell [Aeschlimann/ pg 917/ C].1
References:
Aeschlimann JR. The role of multidrug efflux pumps in the antibiotic resistance of Pseudomonas aeruginosa and other Gram-negative bacteria. Pharmacotherapy. 2003;23:916–924.
Nikaido H. Preventing drug access to targets: Cell surface permeability barriers and active efflux in bacteria. Semin Cell Dev Biol ;12:215–23.
Inner Membrane
Efflux Transporter (AcrB)
Nikaido H. Semin Cell Dev Biol. 2001;12:215–23.

10. Eflux pumps

11. Acinetobacter baumannii
Susceptibility: amikacina R cefepime R ceftazidime R tigeciclina R ampicilina/sulb R
meropenem R imipenem R >8 R amikacina R

12. “A.baummanii resistant Island
45 resistant genes for :
Aminoglicosydes
Tetracyclin
Cotrimoxazol
Chloramphenicol
Betalactams (VEB-1, OXA-10)
Sulfonamides
These genes belongued to other Gram (-) bacteria like Pseudomonas sp., Salmonella sp., y E. coli.
AbaR1-10!
Fournier, P. E., D. Vallenet, V. Barbe, S. Audic, H. Ogata, L. Poirel, H.Richet, C. Robert, S. Mangenot, C. Abergel, P. Nordmann, J. Weissenbach,D. Raoult, and J. M. Claverie Comparative genomics of multidrug resistance in Acinetobacter baumannii. PLoS Genet. 2:e7.

13. BETA-LACTAMASE CLASSIFICATION Bush – Jacoby - Medeiros
Group Characteristics Representative Enzymes 1 2a 2b
2be
2br 2c 2d 2e 2f 3 4
Cafalosporinases not- inhibited by Clav acid ( C.A)
Penicilinases inhibted by C.A
Broad spectrum B-lactamases inh by C.A
Extended spectrum B-lactamases inhibited by C.A (ESBLs)
Broad spectrum B-lactamases with reduced Af to Inhs (IRTs)
Carbenicilinases inhibited by C.A
Cloxacilinases inhibited by C.A
Cefalosporinases inhibited by C.A
Carbapenemases non metallo b -lactamases
Metallo b – lactamases
Penicilinases not- inhibited by C.A
AmpC
PC1 (S. aureus)
TEM-1, TEM-2, SHV-1
TEM , SHV-2 - 6
TEM , TRC-1
PSE-1, CARB-3
OXA-1, PSE-2
P. Vulgaris
IMI-1, NMC-A, Sme-1
L1 (Sten. maltophilia)
P. cepacia

14. Enterobacter cloacae Amikacina S Ampicilina R Cefazolina S
Ceftazidime S
Cefepime S
Ceftriaxone S
Gentamicin S
Levofloxacin S

15. Amp C B-lactamases Hydrolyze 3rd generation cefalosphorins and
B-lactamase Inhibitors
A Aereomonas/ A.baumannii
M Morganella
P Providencia/P.aeruginosa
C Citrobacter freundii
● E Enterobacter
● S Serratia

16. AmpC de-repression
Lister PD. et al. Clinical microbiology reviews, Oct. 2009, p. 582–610

17. Selecting Enterobacter R to 3rd gen cephs during Rx
Rx included
Selection
(49)
No selection (428)
Pen/ampicillin
44.9%
43.05
1 gen cephs
24.5%
26.9%
3 gen cephs
63.3%
30.4% (P<0.001)
Imipenem
16.3%
8.2% (NS)
Pip/tazo
10.2%
8.2%
Amp/sulb
26.5%
28.5%
Kaye et al. AAC 2001, 45, 2628

18. Selecting Enterobacter R to 3rd gen cephs during Rx
Kaye et al. AAC 2001, 45, 2628; Cosgrove et al. Arch Intern Med 2002, 162, 185

19. Prevalence of de-represion
Varies with time, place and patient
30-40% of Enterobacter spp & C. freundii
10-20% of M. morganii
● There is no way to predict it from the micro lab
● Phenotype : resistance to 3rd gen cephalosporins and Pip/taz, but S to Cefepime and Carbapenems.

20. Klebsiella pneumoniae
Amikacin S
Ampicillin R
Cefazolin R
Ceftazidime S/R
Cefepime S/R
Ceftriaxone R/S
Ertapenem S
Meropenem S
Levofloxacin S
Pip/tazobactam S/R

21. ESBLs B-lactamases 3rd gen cephalosporins and Aztreonam.
● Hydrolyze
3rd gen cephalosporins and Aztreonam.
● Lab can diagnose an ESBL in Klebsiella, E.coli & Proteus but they can be found in any Gram (-)
● Plasmids can carry genes that confer resistance to :
Chloramfenicol
Aminoglycosides
Tetracyclines
TMP – S
Quinolones.

22. ESBLs : plasmid mediated
Transposons:
(mobile genetic elements)
Transferable Plasmid
carries genetic information,
including codes for
various resistance
factors
beta-lactamase resistance
genes coding for
Figure developed from: Livermore/ pg S13/ A; pg s14/ A; Jacoby; pg 1700/ A
This slide illustrates plasmid-mediated antibiotic resistance, which includes genes for ESBL production/regulation and aminoglycoside modification (multiresistance) [Livermore/ pg S13/ A; pg S14/ A].2
Resistance factors other than those for ESBLs may reside on the transferable plasmids observed in enterobacteriaceae. The plasmids tend to be large (80 kb) and carry variable resistance to different antimicrobial agents [Jacoby/ pg 1700/ A]. Plasmids encoding ESBLs may carry numerous antibiotic-resistance genes [Jacoby/ 1700/ A].1
Transposons are genetic elements that may encode ESBL production or other resistance and can replicate and reinsert into other plasmids or a host chromosome, thus spreading resistance [Livermore/ pg S13/ A].2
References:
1. Jacoby GA, Medeiros AA. More extended-spectrum b-lactamases. Antimicrob Agents Chemother ;35:1697–1704.
2. Livermore DM. Bacterial resistance: Origins, epidemiology, and impact. Clin Infect Dis ;36:S11–S23.
1. Jacoby. Antimicrob Agents Chemother
2. Livermore DM. Clin Infect Dis. 2003;36(Suppl 1):S11–23.

23. Mortality among 60 patients with ESBL producing Klebsiella Bacteremia
Paterson DL. CID 2004

24. Carbapenamases Enzyme Classification Most Common Bacteria Class A
(serine-b-lactamase)
KPC, SME, IMI, NMC, GES
Enterobacteriaceae
(less common in P. aeruginosa)
Class B
(metallo-b-lactamase)
IMP, VIM, GIM, SPM
NDM
P. aeruginosa
Enterobacteriacea
Acinetobacter spp.
Class D
OXA
( less common in Enterobacteriacea)

25. Klebsiella pneumoniae
Amikacin S/R
Tygecicline S/R
Fosfomycin S/R
Ceftazidime R
Ceftaz/avibactam S
Ceftriaxone R
Ertapenem R
Meropenem S/R
Levofloxacin S/R
Pip/tazobactam R

26. Modified Hodge Test
POSITIVE
NEGATIVE

27. Observational Studies Associating Therapy and Clinical Outcome Bacteremia Caused by KPC-Producing Klebsiella pneumoniae
Study
(Year)
Treatment (# of patients)
Mortality
Daikos et al.
( )
Greece
Combination Therapy (n=103)
Carbapenem-containing (n=31)
Carbapenem-sparing (n=72)
Monotherapy (n=72)
27.2%a
19.3%
30.6%
44.4%a
For carbapenem-containing combinations; mortality higher for MIC >8 mg/L (35.5%) vs 8 mg/L (19.3%)
a 28-day mortality rate; P = 0.003
Colistin and meropenem generally given at higher doses (270 mg/day and 6 g/day, respectively)
Daikos GL, et al. Antimicrob Agents Chemother 2014; 58:

28. In Summary
The interpretation of the mechanisms of resistance in an Antibiogram will help the clinician to :
1. Select the righ antibiotic
2. Decrease the patients morbility and mortality
3. Build Antibiotic Guidelines according to the
hospital data
4. Implement strategies through the ID committee
in order to contain the MDR bacteria
5. Decrease hospital costs