AST and Tests for Resistance

This week in lab a number of tests were performed to determine the susceptibility and resistance of Enterobacter cloacae, an unknown gram-positive cocci, Streptococcus pneumoniae, MRSA, and M. catarrhalis. Antimicrobial susceptibility tests are done to determine what antimicrobial agents are effective against isolated pathogens in vitro. Results of antimicrobial susceptibility tests guide the clinician in initial and later treatment of the patient. They are also used for epidemiological purposes in detecting outbreaks of hospital acquired infections and in locating resistant strains in the community.

An agar disc diffusion susceptibility test was performed on Enterobacter cloacae. The surface of a Mueller Hinton agar plate was inoculated with a standardized suspension of the organism in three directions so that the entire surface was completely covered. Eight antibiotic disks were pressed on the surface of the plate and it was incubated overnight at 35 C. The antimicrobial agents diffuse from the disks into the medium in a circle. As the distance from the disk increases there is a logarithmic reduction in antibiotic concentration, creating a gradient of drug concentrations in the agar medium surrounding each disk. The bacteria inoculated on the surface that are not inhibited by the antibiotic grow flush with the disk and no zone of inhibition is evident. In areas where the concentration of drug is inhibitory no growth occurs.

An MIC of an unknown gram positive cocci was also performed. Various antimicrobial agents are diluted in Mueller-Hinton broth supplemented with calcium and magnesium and dried in the microwells. After inoculation and rehydration with a standardized suspension of organism and incubation at 35 C for a minimum of 16 hours, the MIC for the test organism is determined by observing the lowest concentration showing inhibition of growth.

An E test on Streptococcus pneumoniae was also performed. The E test (also known as the Gradinet Diffusion Method) is based on the same principle as the disk diffusion method. It is an in vitro method for quantitative antimicrobial susceptibility testing whereby a preformed antimicrobial gradient from a plastic-coated strip diffuses into an agar medium inoculated with the test organism. The MIC is read directly from a scale on the top of the strip at a point where the ellipse of organism growth inhibition intercepts the strip.

Selective and differential medium for qualitative direct detection of methicillin resistant Staphylococcus aureus (MRSA) was also determined using CHROMagar MRSA. The medium permits the direct detection and identification of MRSA through the incorporation of specific chromogenic substrates and cefoxitin. MRSA stains will grow in the presence of cefoxitin and produce mauve colored colonies resulting from hydrolysis of the chromogenic substrate. Additional selective agents are incorporated for the suppression of gram negative organisms, yeast and some gram-positive cocci. Bacteria other than MRSA may utilize other chromogenic substrates in the medium resulting in blue to blue/green colored colonies or if no chromogenic substrates are utilized, colonies appear white or colorless. 

A Nitrocefin Disk for beta-lactamase on M. cattarrhalis was also performed. Nitrocefin disks are used for the rapid detection of β-lactamase enzymes in isolated colonies of Neisseria gonorrhoeae, Moraxella catarrhalis, Staphylococcus spp., Haemophilus influenzae and anaerobic bacteria. A positive beta-lactamase result is recorded when the Nitrocefin Disk changes in color from its original yellow to orange or red. Most positive bacterial strains will produce a color change within 5 minutes. Some staphylococci, however, may take up to 60 minutes for a positive result. A positive beta-lactamase result predicts the following:

1. Resistance to penicillin, ampicillin and amoxicillin among Haemophilus spp., N. gonorrhoeae and M. catarrhalis.

2. Resistance to penicillin, as well as acylamino-, carboxy-, and uriedo-penicillins among staphylococci and enterococci.

A negative beta-lactamase result is recorded when the Nitrocef Disk™ remains yellow in color. A negative result does not rule out resistance due to other mechanisms.  

Below is a link to the University of Pennsylvania's Medical Center explanation of antimicrobial susceptibility testing. Click on the links within the site for more detailed information:


http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/amt.html