Small Ruminant Antimicrobial Decision-Making: Interpreting Susceptibility Results
ACVIM 2008
Virginia R. Fajt, DVM, PhD, DACVCP
College Station, TX, USA

What is Susceptibility and Resistance?

These are two related terms to describe the same concept, at least when it comes to use of the terms by clinicians: the concentration of antimicrobial required to inhibit growth of an isolate of bacteria as it relates to the likelihood of clinical success. "Susceptible" and "resistant" are qualitative terms used to simplify the results of tests of growth inhibition. Problems with interpretation can occur when the definitions underlying the qualitative terms are not universally agreed upon (you say the sun has risen when the first ray of light is visible and I say it is when the entire sun is above the horizon) or when different thresholds are used by the individual that performs the test and the individual that uses the results of the test.

Inherent vs. Acquired Resistance

Differentiating inherent and acquired resistance is important from a clinical and epidemiological standpoint, since the concern for resistance associated with treatment failure is generally speaking due to bacteria acquiring resistance genes from other bacteria, rather than a gene that has always been present in a particular bacterial species.

Inherent resistance includes mechanisms such as the gram-negative cell wall, which is thick enough to prevent certain drugs from entering the cell, or the oxygen-dependent nature of aminoglycosides crossing the bacterial cell wall, which means that anaerobes are not inhibited by aminoglycosides.

Acquired resistance is most often generated by the acquisition of new bacterial DNA by various mechanisms, including transfer of plasmids (extra-chromosomal DNA) between a resistant and a susceptible organism, or transfer of an integron or other type of moveable gene between a resistant and a susceptible organism. Transfer of resistance can occur between bacteria of the same species, but also between bacteria of different species and even of different genera. The significance of this transfer is that the bacteria containing these resistance genes may be selectively targeted for survival by the presence of an antimicrobial, and the resistance DNA is then carried on the next generation or is transferred to other bacteria. This DNA may encode for an enzyme that destroys the antimicrobial, which changes the conformation of the cell wall so that the antimicrobial cannot bind, or which changes the binding site on a ribosome so the antimicrobial can no longer interfere with protein synthesis.

How Can We Determine Susceptibility or Resistance?

Since we have described resistance as requiring more antimicrobial than can be delivered to the infection site, we need a way to determine how much of a particular antimicrobial is required to inhibit the growth of a particular organism. We are all familiar with susceptibility testing as performed by most clinical microbiology labs, but it is useful to briefly review these tests.

Types of Susceptibility TESTING

The two major types of susceptibility testing performed by veterinary diagnostic laboratories are disk diffusion and broth microdilution. Disk diffusion testing uses paper disks containing known quantities of antimicrobials, and the zone around which no growth of bacteria occurs correlates with a particular range of antimicrobial concentrations. The correlation between zone and MIC is a qualitative rather than a quantitative one: zones of inhibition do not linearly correspond to minimum inhibitory concentrations of antimicrobial.

Broth microdilution testing is used to characterize the quantity of antimicrobial required to inhibit bacterial growth. Varying concentrations of antimicrobial are mixed with the broth used to grow bacterial isolates, and the lowest concentration which demonstrates no growth is the MIC. This type of testing is usual performed with 96-well plates so multiple drugs can be tested or even more than one isolate can be tested on a plate. There are of course practical limitations to the number of concentrations for any single antimicrobial, and to the number of antimicrobials that can be tested, so concentrations which are clustered around the breakpoint are generally selected, with the idea that these concentrations are also actually clinically achievable in the animal.

What is a Breakpoint and How Are They Determined?

Are there Different Types or Categories of Breakpoints?

The purpose of the breakpoint is to provide a cutoff or threshold for categorizing organisms with different phenotypes. The phenotypes practicing veterinarians are interested in identifying are those that might be difficult to eliminate in clinical infections because they require increased concentrations of antimicrobials to inhibit growth. However, other groups may be interested in other cutoffs, such as epidemiologic cutoffs, whereby populations of organisms could be evaluated over time to watch for significant changes in susceptibility. Different cutoffs might also be used to evaluate whether the majority of isolates remain wild-type, or whether acquired resistance is penetrating a population. In essence, the breakpoints discussed in this document are clinical breakpoints, related to predicting clinical outcome of antimicrobial therapy for bacterial disease.

How are Breakpoints Developed?

Through a multi-step process, data are gathered and/or generated which allow the Clinical Laboratory Standards Institute Veterinary Antimicrobial Susceptibility Testing Subcommittee (CLSI VAST) to select appropriate breakpoints. (The CLSI is an international, interdisciplinary, nonprofit, standards-developing, and educational organization that promotes the development and use of voluntary consensus standards and guidelines within the health care community.) These data include pharmacokinetics of the drug in question, pharmacodynamics of the drug (how does it work best), and MIC data for at least 100 different isolates of the bacterial species for which the breakpoints will be valid.

Are There Any Breakpoints Validated for Small Ruminants?

No breakpoints have been validated for small ruminants. This means that the breakpoints listed below are not based on pharmacokinetic data from small ruminants, nor from bacterial population MIC data from small ruminant pathogens.

CLSI Example Breakpoints

A few of the drugs commonly used in food-producing animals are included in the table below, which is excerpted from the CLSI document "Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals," (M31-A2). Recognizing that there are no breakpoints validated for small ruminants will help the clinician understand the importance of reviewing these breakpoints when looking at susceptibility results from the laboratory. Knowing that an "S" from the lab actually means that the organism is inhibited at the antimicrobial concentration listed below allows the veterinarian to make dose adjustments that would result in the appropriate drug concentrations (see other proceedings by this author for more information on regimen design).

Note that in the table below, the gray shaded rows in the column are breakpoints which were imported from human susceptibility testing standards; the non-shaded areas are breakpoints which have been validated by the CLSI VAST. These are only a few examples of the drug-pathogen combinations that are listed in the CLSI document, available at http://www.clsi.org.

 

MIC Breakpoint(µg/ml)

Antimicrobial Agent

S

I

R

Ceftiofur

Bovine (Respiratory Disease)

<2

4

>8

 

Mannheimia haemolytica

     

Pasteurella multocida

     

Histophilus somni

     

Swine (Respiratory Disease)

<2

4

>8

 

Actinobacillus pleuropneumoniae

     

Pasteurella multocida

     

Salmonella choleraesuis

<0.25

   

Equine (Respiratory Disease)

     
 

Streptococcus equi subsp.

     

zooepidemicus

     

Florfenicol

Bovine (Respiratory Disease)

<2

4

>8

 

Mannheimia haemolytica

     

Pasteurella multocida

     

Histophilus somni

     

Swine (Respiratory Disease)

<2

4

>8

 

Actinobacillus pleuropneumoniae

     

Bordetella bronchiseptica

     

Pasteurella multocida

     

Streptococcus suis

     

Salmonella cholerasuis

<4

 

>16

Penicillin

Staphylococci

<0.12

 

>0.25

Enterococci

<8

 

>16

Streptococci (not S. pneumoniae)

     
 

viridans group

<0.12

 

>4

beta-hemolytic group

<0.12

   

Listeria spp.

<2

   

Pirlimycin

Bovine mastitis

<2

 

>4

 

Staphylococcus aureus

     

Streptococcus agalactiae

     

Streptococcus dysgalactiae

     

Streptococcus uberis

     

Sulfisoxazole

<256

 

>512

Tetracycline

Organisms other than streptococci

<4

8

>16

Streptococcus pneumoniae

<2

4

>8

Tilmicosin

Bovine (Respiratory Disease)

<8

16

>32

 

Mannheimia haemolytica

     

Pasteurella multocida

     

Swine (Respiratory Disease)

<16

 

>32

 

Pasteurella multocida

     

Actinobacillus pleuropneumoniae

     

Summary of Common Pitfalls in Interpreting Susceptibility Results

1.  A result of "S" or "susceptible" from a diagnostic lab for a pathogen isolated from a small ruminant should be used with care. Recall that there are no validated breakpoints for pathogen-drug combinations for sheep or goats, and there are demonstrated differences between cattle and small ruminant pharmacokinetics as well as among susceptibility patterns from cattle and small ruminant pathogens.

2.  Susceptibility test results for enteric pathogens are essentially meaningless, since there are NO validated breakpoints for any pathogens or antimicrobials. There may be clinical trial data that show therapeutic efficacy for a particular pathogen with a particular drug, but there are few or no published clinical efficacy data that correlate clinical efficacy, dose, and drug concentration at the site of infection. Culture results may be useful for identifying pathogens, but susceptibility test results are not useful.

3.  Because there are few published susceptibility data in pathogens of small ruminants, veterinarians are encouraged to do as much susceptibility testing as possible, whether you test individual cases (e.g., non-responsive cases, recurring cases, valuable animals, etc.) or whether you attempt to periodically gather data in your practice for the purposes of collecting your own population data.

References

1.  Clinical Laboratory Standards Institute, "Performance Standards for Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals; Approved Standard--Second Edition," M31-A2, 2005.

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Virginia Fajt, DVM, PhD, DACVCP
Texas A&M University
College Station, TX


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