Mechanisms of Aminoglycoside Resistance

There are three mechanisms of aminoglycoside resistance: reduced uptake or decreased cell permeability, alterations at the ribosomal binding sites, or production of aminoglycoside modifying enzymes.

Reduced uptake or decreased cell permeability

Some strains of Pseudomonas aeruginosa and other gram-negative bacilli exhibit aminoglycoside resistance due to a transport defect or membrane impermeabilization.1, 4  This mechanism is likely chromosomally mediated and results in cross-reactivity to all aminoglycosides.1,4  The level of resistance that is seen is moderate (i.e. intermediate susceptibility).1

Altered Ribosome Binding Sites

Mutations at the site of aminoglycoside attachment may interfere with ribosomal binding.  Resistance to streptomycin can occur by this mechanism since this agent binds to a single site on the 30S subunit of the ribosome.  Resistance to the other aminoglycosides by this mechanism is uncommon since they bind to multiple sites on both ribosomal subunits and high-level resistance cannot be selected by a single step.4

Enzymatic Modification

      Enzymatic modification is the most common type of aminoglycoside resistance. Over 50 different enzymes have been identified.5  Enzymatic modification results in high-level resistance.4  The genes encoding for aminoglycoside modifying enzymes are usually found on plasmids and transposons.  Most enzyme-mediated resistance in gram-negative bacilli is due to multiple genes.  It is hypothesized that the enzymes are derived from organisms that make the aminoglycoside or from the mutation of genes that encode the enzymes involved in cellular respiration.2

      There are three types of aminoglycoside modifying enzymes: 5

1.      N-Acetyltransferases (AAC) – catalyzes acetyl CoA-dependent acetylation of an amino group

2.      O-Adenyltransferases (ANT) – catalyzes ATP-dependent adenylation of hydroxyl group

3.      O-Phosphotransferases (APH) – catalyzes ATP-dependent phosphorylation of a hydroxyl group

  

Genes Encoding for Aminoglycoside Resistance 6

The following is a table of common genes encoding for aminoglycoside modifying enzymes (this table is not all inclusive).

The nomenclature is defined as follows: AAC, ANT, or APH for the type of enzymatic modification, followed by a number in parentheses designating the site of modification. 

The Roman numerals and letters that follow stand for unique resistance profiles and protein designations, respectively.

Enzyme

Genes

Selected Aminoglycoside Substrates

Comments

Acetylation

     

AAC(3)-I

aac(3)-Ia

aac(3)-Ib

Gm

 

AAC(3)-II

aac(3)-Iia

aac(3)-Iib

aac(3)-Iic

Gm, Tob

 

AAC(3)-III

aac(3)-IIIa

aac(3)-IIIb

aac(3)-IIIc

Gm, Tob, Km, Neo, Prm

Commonly found in Pseudomonas spp.

Rarely seen in Enterobacteriaceae

AAC(3)-IV

aac(3)-Iva

Gm, Tob

Commonly found in Salmonella spp.

AAC(3)-VI

aac(3)-Via

Gm

Resistance to Tob and Km not conferred; however a low-level of enzymatic activity has been detected.

Rare among Enterobacteriaceae

AAC(6’)-I

aac(6’)-Ia

aac(6’)-Ib

aac(6’)-Ic

aac(6’)-Id

aac(6’)-Ie

aac(6’)-If

aac(6’)-Ig

aac(6’)-Ih

aac(6’)-Ii

Tob, Amk

AAC(6’)-II

aac(6’)-Iia

aac(6’)-Iib

Gm, Tob

Observed only in P. aeruginosa

AAC(6’)-APH(2”)

aac(6’)-aph(2”)

Gm, Tob, Amk

Bifunctional enzyme thought to be restricted to gram positive bacteria. (staphylococci and enterococci)

AAC(2’-I)

aac(2’)-Ia

Gm, Tob

Adenylylation

   

ANT(2”)-I

ant(2”)-Ia

ant(2”)-Ib

ant(2”)-Ic

Gm, Tob, Km

Widespread among all gram-negative bacteria

ANT(3”)-I

ant(3”)-Ia

Sm, Spcm

ANT(4’)-I

ant(4’)-Ia

Tob, Amk

ANT(4’)-II

ant(4’)-Iia

Tob, Amk

ANT(6)-I

ant(6)-Ia

Sm

Found in gram-positive organisms

Phosphorylation

   

APH(2”)-I

aph(2”)-Ia

Gm, Tob, Amk

APH(3’)-I

aph(3’)-Ia

aph(3’)-Ib

aph(3’)-Ic

Km, Neo, Prm

APH(3’)-II

aph(3’)-Iia

Km, Neo, Prm, GmB

APH(3’)-III

aph(3’)-IIIa

Km, Neo, Prm, Amk, GmB

Commonly found in S. aureus and E. faecalis

APH(3’)-IV

aph(3’)-Iva

Km, Neo, Prm

APH(3’)-V

aph(3’)-Va

aph(3’)-Vb

aph(3’)-Vc

Neo, Prm

APH(3’)-VI

aph(3’)-Via

aph(3’)-Vib

Km, Neo, Prm, Amk, GmB

Primarily isolated from Acinetobacter spp.

APH(3’)-VII

aph(3’)VIIa

Km, Neo

Cloned from Campylobacter jejuni

APH(3”)-I

aph(3”)-Ia

aph(3”)-Ib

Sm

Cloned from Streptomyces griseus

APH(6)-I

aph(6)-Ia

aph(6)-Ib

aph(6)-Ic

aph(6)-Id

Sm

Cloned from Streptomyces spp.

This table was adapted from: Shaw KJ, Rather PN, Hare RS, et al. Molecular Genetics of Aminoglycoside Resistance Genes and Familial Relationships of the Aminoglycoside-Modifying Enzymes.  Microbiological Reviews 1993;57:138-63.

Abbreviation key: Amk, amikacin; Gm; gentamicin, GmB, gentamicin B; Km, kanamycin; Neo, neomycin; Pm, paromycin; Spcm, spetinomycin; Sm, streptomycin; Tob, tobramycin

 

 

Phenotypic Characterization of Aminoglycoside Resistance Mechanisms

Overview

A large and diverse population of aminoglycoside-modifying enzymes exist and act at virtually every susceptible position on aminoglycoside structures. 1  By testing the susceptibility of isolates against a range of clinically available and experimental aminoglycosides, a pattern of resistance emerges that is unique to a specific enzyme.1  This method has been referred as interpretative reading.7

-Phenotypic Characterization by Interpretative Reading-

Examples of aminoglycoside resistance phenotypes
of Enterobacteriaceae spp.,including E. Coli
(excluding Serratia spp. and Klebsiella spp.)

Phenotype

classical

AAC(3)I

AAC(3)II

AAC(3)IV

AAC(6’)

ANT(2’)

APH(3’)

Gentamicin

S

R

R

R

S/r

R

S

Netilmicin

S

S

R

R

R

S

S

Tobramycin

S

S

R

R

R

R

S

Amikacin

S

S

S

S

R

S

S

Kanamycin

S

S

R

r

R

R

R

Neomycn

S

S

S

R

R

S

R

classical= historic phenotype of the species, without acquired resistance
S=susceptible, R=resistant, r=reduced zones but likely to remain susceptible at standard breakpoints
Adapted from: Livermore DM, Winstanley TG, Shannon, KP. Interpretative reading: recognizing the unusual and inferring resistance mechanisms from resistance phenotypes. Journal of Antimicrobial Chemotherapy. 2001;48(Suppl S1), 87-102.

               

Examples of aminoglycoside resistance of Enterococcus faecalis

Phenotype

classical*

ANT(4’)(4”)I

APH(2”)/
AAC(6’)

APH(3’)

AAC(3’)III

Gentamicin

R

R

HLR

R

R

Netilmicin

R

R

R

R

R

Tobramycin

R

HLR

HLR

R

R

Amikacin

R

HLR

R

R

HLR

Kanamycin

R

HLR

HLR

HLR

HLR

Neomycn

R

R

R

HLR

HLR

* intrinsic low-level resistance, classical = historic phenotype of the species, without acquired resistance
R=resistance, HLR= high-level resistance
Adapted from: Livermore DM, Winstanley TG, Shannon, KP. Interpretative reading: recognizing the unusual and inferring resistance mechanisms from resistance phenotypes. Journal of Antimicrobial Chemotherapy. 2001;48(Suppl S1), 87-102.

Prevalence of  Aminoglycoside Resistance by Geographic Region

Gentamicin Resistance among Enterococci

Enterococci are an important cause of urinary tract infections and are becoming an increasingly important cause of bloodstream infections.  The penicillin or vancomycin and gentamicin combination is synergistically bactericidal against enterococcal strains that exhibit low-level resistance to gentamicin, while strains that exhibit high-level resistance are unaffected. 

Low level resistance to all aminoglycosides is due to limited drug uptake by enterococci, and is most likely a result of their facultative anaerobic metabolism.  The acquisition of aminoglycoside resistance genes by enterococci has resulted in high level resistance with MIC's usually > 2000 mcg/ml.  The bifunctional gene aac(6')-Ie-aph(2")-Ia encodes the aminoglycoside modifying enzyme AAC(6')-Ie-APH(2")-Ia which confers resistance to all of the aminoglycosides with the exception of streptomycin.  This is, clinically, the most important enzyme.  Other genes: aph(2")-Ib, aph(2")-Ic, and aph(2")-Id  encode for aminoglycoside modifying enzymes that confer resistance to gentamicin.  Some enterococci may possess multiple genes.  Resistance to streptomycin can also be enzymatic by production of AAC(6')-Ia or ANT(3")-Ia, or it can be by single-step ribosomal protein mutation.8

Prevalence of Gentamicin Resistance of Enterococci in Various Regions

Country or Region

Years

# Isolates

% Gent Resistance

(High-level)

Reference

All sites of infection (blood, respiratory, wound, urine)

United States Philadelphia*

2000

2001

175

205

52

69

This web site

United States - Nationally

1997

1998

1999

2400

29

28

31

CID 2001:32; S133

Canada

1997

1998

1999

509

40

40

35

Latin America

1997

1998

1999

367

32

14

16

Europe

1997

1998

1999

1201

29

28

26

Asia-Pacific

1998

1999

331

36

30

                                *blood isolates only

Aminoglycoside Resistance in Pseudomonas aeruginosa

Aminoglycoside antibiotics have been used for many years against Pseudomonas aeruginosa.  The aminoglycosides have been shown to be synergistic with beta-lactam antibiotics and are commonly used in combination with these agents empirically and in the treatment of Pseudomonal infections.  Although resistance to the aminoglycosides is increasing, they continue to play an important role in the treatment of these infections.

There are several mechanisms of resistance reported with Pseudomonas aeruginosa.  One mechanism is increased impermeability across the cell wall.  This has been evidenced in strains that lack modifying enzymes and do not exhibit cross-resistance with other classes of antibiotics.  The actual mechanism for this type of resistance is not known.  More recent data suggest that up-regulation of the efflux system MexXY-OprM affects the aminoglycoside antibiotics.  Lastly, Pseudomonas aeruginosa isolates have been shown to contain aminoglycoside modifying enzymes.  Those isolated include: AAC(6')-I, APH(2"), APH(3')-VI, and AAC(3)-II, and there may be more.9,10

Country or Region

Years

# Isolates

% Gent Resistance

% Tobra Resistance

% Amik

Resistance

Reference

United States –

Philadelphia, UPHSa

2000

2001

436

453

27

26

14

12

15b

12b

This web site

United States – Nationally

1997

1998

1999

~2474

not reported

8.9

7.3

7.8

5

5.2

3.4

CID 2001:32; S146

Latin America

1997

1998

1999

~1093

not reported

31.9

35.1

35.8

22.4

26.7

30.5

Canada

1997

1998

1999

~582

not reported

8.6

6.6

5.8

8.6

4.2

2.2

Asia-Pacific

1998

1999

~755

not reported

11.5

10.1

5.8

4.2

Europe

1997

1998

1999

~1656

not reported

23.7

21.7

31.6

11

13.2

21.1

France

4/97 – 4/98

140

28

29

1

Eur J Clin Microbiol Infect Dis 1999:18; 414

Germany

4/97-4/98

31

10

10

0

Italy

4/97- 4/98

75

33

33

10

Poland

4/97- 4/98

28

39

39

14

Spain

4/97- 4/98

100

10

4

2

                                         a includes blood, non-urinary, and urinary isolates

                                         b blood and non-urinary isolates only

Summary/Practical Implications of Aminoglycoside Resistance

With the exceptions of P. aeruginosa and Enterococcus spp., the incidence of aminoglycoside resistance remains low.  Multiple mechanisms for aminoglycoside resistance exist.  Enzymatic modification, the most common mechanism, involves so many different enzymes and phenotypes that resistance patterns are difficult to predict without complicated methods such as interpretative reading.  Many clinical microbiology laboratories utilize these methods to modify the institution’s antibiogram.   Practical use of these methods for clinicians means referring to the institution’s antibiogram when selecting aminoglycosides for empiric use and to patient specific culture and susceptibility data for definitive use.

References

  1. Mingeot-Leclercq MP, Glupczynski Y, and Tulkens PM. Aminoglycosides: Activity and Resistance. Antimicrobial Agents and Chemotherapy 1999;43:727-37.
  1. Gilbert D. Aminoglycosides. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 5th ed. Philadelphia: Churchill Livingstone, 2000:307-336.
  1. Opal S, Mayer K, and Medeiros A.  Mechanisms of Bacterial Antibiotic Resistance. In:   Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 5th ed. Philadelphia: Churchill Livingstone, 2000:236-252.
  1. Kucers A, Crowe S, Grayson ML, and Hoy J, eds.   The Use of Antibiotics: A Clinical Review of Antibacterial, Antifungal, and Antiviral Drugs. 5th ed. Oxford: Butterworth Heinemann, 1997:452-457.
  1. Davies J and Wright G. Bacterial Resistance to Aminoglycoside Antibiotics. Trends in Microbiology 1997;5:234-39.
  1. Shaw KJ, Rather PN, Hare RS, et al. Molecular Genetics of Aminoglycoside Resistance Genes and Familial Relationships of the Aminoglycoside-Modifying Enzymes.  Microbiological Reviews 1993;57:138-63. 
  1. Livermore DM, Winstanley TG, Shannon, KP. Interpretative reading: recognizing the unusual and inferring resistance mechanisms from resistance phenotypes. Journal of Antimicrobial Chemotherapy. 2001;48(Suppl S1), 87-102.
  1. Chow JW.  Aminoglycoside resistance in enterococci.  Clinical Infectious Diseases.  2000;31:586-9.
  1. Livermore DM.; Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare?  Clinical Infectious Diseases. 2002;34:634-40.
  1. Kettner M, Milosovic P, Hletkova M, Kallova J.  Incidence and mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa serotype 011; isolates.; Infection. 1995;23(6): 380-3.

Written by: Catherine Barnhart, PharmD, Ronald Campbell, PharmD, Lori Ann LaRosa, PharmD, Ann Marie Marr, PharmD, Amy Morgan, PharmD, Derek VanBerkom, PharmD, October 2002