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

Antimicrobial Susceptibility Testing

Susceptibility testing involves testing pathogens against antimicrobial agents to determine if the bacteria express resistance to agents that could potentially be used in treatment. The microdilution method of measuring antimicrobial susceptibility is commonly employed in the clinical laboratory. The procedure begins by preparing a standard suspension of the organism and dispensing it into the seed tray. A specialized inoculator device with prongs dispenses 0.01 mL of inoculum into wells of microdilution trays containing various concentrations of antimicrobial agents. The plates are continuously monitored by an automated system.The instrument detects the growth in the wells by spectrophotometry (or fluorometric analysis) by comparing wells with antimicrobics to control wells without antimicrobics. 

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The antimicrobial agents that are chosen for testing against a particular bacterial isolate are referred to as the antimicrobial battery. Antimicrobials to which the organism is intrinsically resistant are routinely excluded from the test battery. Similarly, certain antimicrobials were specifically developed for use against particular organisms but not others and should be included in the appropriate battery. If resistance to a particular agent is common, the utility of the agent may be sufficiently limited so that routine testing is not warranted and only more potent antimicrobials are included in the test battery. Conversely, more potent agents may not need to be in the test battery is susceptibility to less potent agents is highly prevalent. 

Further considerations about antibiotics may be employed against particular organism groups. The most common individual testing batteries are considered for Enterobacteriaceae, P. aeruginosa, Acenitobacter spp, Staphylococcus spp, Enterococcus spp, Streptococcus spp, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae. Below is a link to an example of specific information for susceptibility testing of S. pneumoniae.

http://www.cdc.gov/abcs/reports-findings/downloads/lab-methods.pdf

Pasteurella multocida

Pasteurella multocida is a nonmotile gram negative coccobacilli. The organism grows best
 at 37 C and exhibits both oxidative and fermentive metabolism. P. multocida produces grey, rough, viscous, non-hemolytic colonies on blood agar and no growth on MacConkey agar. They are also catalase and oxidase positive with a characteristic "mousy"odor.

Pasteurella species are commonly isolated pathogens in most animal bites, especially in dog- and cat-related injuries. These injuries can be aggressive, with skin manifestations typically appearing within 24 hours following a bite that exhibit a rapidly progressive soft-tissue inflammation.

If left untreated Pasteurella infections can lead to cellulitis and can spread to cause serious systemic infection. Most reported cases of Pasteurella infections  involve skin and soft tissue with occasional development of regional lymphadenopathy. Beyond skin and soft tissue, other sites of infection are uncommon and have been the subject of individual case reports or small case series (they include bone and joint infections, CNS infections, septicemia, endocarditis, respiratory tract infections and intra-abdominal infections).

For more information about P. multocida or for a quick reference for other organisms visit the following link:

http://www.amrita.ac.in/bioprojects/IndusMicroBio/Micro.php

Armadillos and Leprosy

Mycobacterium leprae is an acid fast Gram-positive bacterium, with a slow doubling time of 14 days. The slow doubling time is due to the restricted intake of nutrients through the pores in the large waxy walls. Mycobacteria, as genus, are typically found in the soil, water and in the air.Leprosy is very specified when it comes to infecting hosts. Its ideal conditions are around 33 degrees C, which is lower than most mammals. Mammals with lower temperatures are better hosts for leprosy. That is why only a few species are known to be carriers of M. leprae. This is also why in humans, leprosy tends to be found primarily at the peripheral nerves. Hands and feet tend to be cooler than the core body temp
erature, providing a more habitable environment for M. leprae.



Mycobacterium leprae is an acid fast Gram-positive bacterium, with a slow doubling time of 14 days. The slow doubling time is due to the restricted intake of nutrients through the pores in the large waxy walls. Mycobacteria, as genus, are typically found in the soil, water and in the air.Leprosy is very specified when it comes to infecting hosts. Its ideal conditions are around 33 degrees C, which is lower than most mammals. Mammals with lower temperatures are better hosts for leprosy. That is why only a few species are known to be carriers of M. leprae. This is also why in humans, leprosy tends to be found primarily at the peripheral nerves. Hands and feet tend to be cooler than the core body temperature, providing a more habitable environment for M. leprae.


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